Method for modifying genome sequence to introduce specific mutation to targeted DNA sequence by base-removal reaction, and molecular complex used therein

ABSTRACT

The present invention provides a method of modifying a targeted site of a double stranded DNA, including a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a selected double stranded DNA and DNA glycosylase with sufficiently low reactivity with a DNA having an unrelaxed double helix structure (unrelaxed DNA) are bonded, with the double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into the targeted site, without cleaving at least one strand of the double stranded DNA in the targeted site.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is the U.S. national phase of InternationalPatent Application No. PCT/JP2015/080958, filed Nov. 2, 2015, whichclaims the benefit of Japanese Patent Application No. 2014-224745, filedon Nov. 4, 2014, which are incorporated by reference in their entiretiesherein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 49,254 bytes ASCII (Text) file named“728697ReplacementSequenceListing.txt,” created Dec. 27, 2018.

TECHNICAL FIELD

The present invention relates to a modification method of a genomesequence, which enables modification of a nucleic acid base in aparticular region of a genome, without cleaving double stranded DNA (nocleavage or single strand cleavage), or inserting a foreign DNAfragment, but utilizing a base excision reaction, and a complex of anucleic acid sequence-recognizing module and DNA glycosylase to be usedtherefor.

BACKGROUND ART

In recent years, genome editing is attracting attention as a techniquefor modifying the object gene and genome region in various species.Conventionally, as a method of genome editing, a method utilizing anartificial nuclease comprising a molecule having a sequence-independentDNA cleavage ability and a molecule having a sequence recognitionability in combination has been proposed (non-patent document 1).

For example, a method of performing recombination at a target gene locusin DNA in a plant cell or insect cell as a host, by using a zinc fingernuclease (ZFN) wherein a zinc finger DNA binding domain and anon-specific DNA cleavage domain are linked (patent document 1), amethod of cleaving or modifying a target gene in a particular nucleotidesequence or a site adjacent thereto by using TALEN wherein atranscription activator-like (TAL) effector which is a DNA bindingmodule that the plant pathogenic bacteria Xanthomonas has, and a DNAendonuclease are linked (patent document 2), a method utilizingCRISPR-Cas9 system wherein DNA sequence CRISPR (Clustered Regularlyinterspaced short palindromic repeats) that functions in an acquiredimmune system possessed by eubacterium and archaebacterium, and nucleaseCas (CRISPR-associated) protein family having an important functionalong with CRISPR are combined (patent document 3) and the like havebeen reported. Furthermore, a method of cleaving a target gene in thevicinity of a particular sequence, by using artificial nuclease whereina PPR protein constituted to recognize a particular nucleotide sequenceby a continuation of PPR motifs each consisting of 35 amino acids andrecognizing one nucleic acid base, and nuclease are linked (patentdocument 4) has also been reported.

These genome editing techniques basically presuppose double stranded DNAbreaks (DSB). However, since they include unexpected genomemodifications, side effects such as strong cytotoxicity, chromosomalrearrangement and the like occur, and they have common problems ofimpaired reliability in gene therapy, extremely small number ofsurviving cells by nucleotide modification, and difficulty in geneticmodification itself in primate ovum and unicellular microorganisms.

On the other hand, as a method of performing nucleotide modificationwithout causing DSB, utilization of DNA glycosylase has been proposed.For example, patent document 5 describes that mutant DNA glycosylasehaving an activity to eliminate a thymine or cytosine base from a singlestranded or double stranded DNA (TDG activity or CDG activity) wasobtained by introducing mutation into human uracil-DNA glycosylase (UDG)and, using the enzyme, the efficiency of mutation induction inEscherichia coli was improved.

Furthermore, patent document 6 and non-patent document 2 describe thattargeted nucleotide modification into a genome region having a Tetoperator (TetO) DNA sequence specifically recognized by TetR is possibleby using a fusion protein of yeast 3-methyladenine-DNA glycosylase(MAGI) and Tet repressor protein (TetR). However, MAGI essentiallyremoves a special DNA base that was injured by alkylation and the like,which is mainly 3-methyladenine, and the ability to remove a base fromnormal bases, particularly base pairs without a mismatch, is extremelylow. Therefore, it is difficult to observe the effect of mutationintroduction by MAGI in normal cells, and a detectable mutation rate hasbeen obtained only by overexpressing MAGI in cells with disrupted genesof base excision repair system. In addition, when the DNA-repair systemis weakened, the mutation rate of the entire genome also increases,which makes practicalization a far goal. However, when a mutant UDGhaving CDG activity to act on a normal cytosine base was used for thissystem instead of MAGI, the efficiency of mutation induction did notincrease (patent document 6, non-patent document 2).

DOCUMENT LIST

Patent Documents

-   patent document 1: JP-B-4968498-   patent document 2: National Publication of International Patent-   Application No. 2013-513389-   patent document 3: National Publication of International Patent-   Application No. 2010-519929-   patent document 4: JP-A-2013-128413-   patent document 5: WO 97/25416-   patent document 6: WO 2014/127287    Non-Patent Documents-   non-patent document 1: Kelvin M Esvelt, Harris H Wang (2013)    Genome-scale engineering for systems and synthetic biology,    Molecular Systems Biology 9: 641-   non-patent document 2: Shwan P. Finney-Manchester, Narendra    Maheshri, (2013) Harnessing mutagenic homologous recombination for    targeted mutagenesis in vivo by TaGTEAM, Nucleic Acids Research    41(9): e99

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a novel method ofgenome editing for modifying a nucleic acid base of a particularsequence of a gene without DSB or insertion of foreign DNA fragment,i.e., by non-cleavage of a double stranded DNA or single strandcleavage, and a complex of a nucleic acid sequence-recognizing moduleand a mutation introducing enzyme therefor.

Means of Solving the Problems

The present inventors have already reported that they have successfullymodified, without DSB, a genome sequence by nucleic acid base conversionin a region containing a particular DNA sequence, by using deaminasethat catalyzes a deamination reaction and linking the enzyme and amolecule having a DNA sequence recognition ability (WO 2015/133554). Inexpectation of affording a mutation introduction tendency different fromthat of deaminase and the like, the present invention is based on anidea of causing a base excision reaction by hydrolysis of N-glycosidicbond of DNA, and then inducing mutation introduction in a repair processof cells. Thus, attempts have been made to use an enzyme having CDGactivity or TDG activity, which is a mutant of yeast mitochondrialuracil-DNA glycosylase (UNG 1), as an enzyme that performs such baseexcision reaction.

The present inventors assumed that one of the reasons for a failure toimprove efficiency of mutation induction by conventional methods (e.g.,patent document 6 and non-patent document 2) even by using mutant UDGhaving CDG activity is that the enzyme causes base excision everywherein the double stranded DNA, which in turn causes high cytotoxicity andmakes it difficult to express the enzyme protein itself. Under suchhypothesis, the present inventors modified a genome sequence by nucleicacid base conversion in a region containing a particular DNA sequence,by introducing a mutation that lowers an action on a DNA having anunrelaxed double helix structure (unrelaxed (double-helical) DNA) into amutant UDG having CDG activity or TDG activity, and linking the mutantenzyme and a molecule having a DNA sequence recognition ability.

To be specific, CRISPR-Cas system (CRISPR-mutant Cas) was used. That is,a DNA encoding an RNA molecule wherein genome specific CRISPR-RNA:crRNA(gRNA) containing a sequence complementary to a target sequence of agene to be modified is linked to an RNA (trans-activating crRNA:tracrRNA) for recruiting Cas protein was produced, a DNA wherein a DNAencoding a mutant Cas protein (dCas) wherein cleavage ability of bothstrands of a double stranded DNA is inactivated and a mutant CDG or TDGgene are linked was produced, and these DNAs were introduced into a hostyeast cell containing a gene to be modified. As a result, mutation couldbe introduced randomly within the range of several hundred nucleotidesof the object gene including the target sequence. Furthermore, mutationcould be introduced extremely efficiently by targeting multiple regionsin the object gene. That is, a host cell introduced with DNA was seededin a nonselective medium, and the sequence of the object gene wasexamined in randomly selected colonies. As a result, introduction ofmutation was confirmed in almost all colonies. The efficiency ofmutation induction was further improved by coexpressing mutant APendonuclease lacking its function as an enzyme of a base excision repairsystem, thereby increasing the frequency of repair errors in an abasicsite (AP site).

It was also confirmed that a system using a heterogenous mutant UDG alsofunctions in yeast. Unexpectedly, UDG derived from vaccinia virus(vvUDG) does not cause cytotoxicity even in the absence of introductionof mutation that lowers the action on an unrelaxed double helixstructure of DNA, and mutant UDG introduced solely with mutations thatchange substrate specificity rather showed higher efficiency of mutationinduction. That is, vvUDG was suggested to be an enzyme having anatively low action on DNA having an unrelaxed double-helix structure.This was also supported by the fact that the mutated sites by vvUDG areconcentrated in a particular base within the target sequence (i.e.,single strand part). In addition, the activity of vvUDG could beincreased by co-expressing A20, which is known as a cofactor.

The present inventors obtained an idea of utilization of split Cas9system as a means to decrease non-specific mutation by UDG, in additionto the introduction of mutation into the enzyme. Split Cas9 is a variantof Cas9, which is designed to function only when N-terminal fragment andC-terminal fragment split from Cas9 protein are associated with gRNA(Nat Biotechnol. 33(2): 139-142 (2015); PNAS 112(10): 2984-2989 (2015)).The present inventors constructed a system expressing split dCas9-mutantUNG1 as one fusion protein consisting of N-terminal fragment ofdCas9-N-terminal fragment of mutant UNG1-C-terminal fragment ofdCas9-C-terminal fragment of mutant UNG1, and a system separatelyexpressing N-terminal fragment of dCas9-C-terminal fragment of mutantUNG1, and N-terminal fragment of mutant UNG1-C-terminal fragment ofdCas9, and then examined the frequency of mutation and non-specificmutation at the target site by using a sequence in the Can1 gene as atarget, as well as cell proliferation. As a result, in any system, thecell survival rate on a nonselective medium is hardly different betweenmutant UNG1 introduced only with mutation (N222D) that imparts CDGactivity, and mutant UNG1 further introduced with mutation (R308C) thatdecreases the action on an unrelaxed double helix structure of DNA, andthe cytotoxicity was markedly reduced by utilization of the splitenzyme. When split enzyme was used, the frequency of non-specificmutation using thialysine resistance as an index was also reducedwithout depending on the presence or absence of R308 mutation. Theefficiency of mutation induction at the original target site ratherdecreased, since addition of the R3080 mutation also decreases the CDGactivity. It was suggested, therefore, a mutation that reduces theaction on an unrelaxed double helix structure of DNA is preferably notadded to UDG when a split enzyme is used.

The present inventor have conducted further studies based on thesefindings and completed the present invention.

Therefore, the present invention is as described below.

[1] A method of modifying a targeted site of a double stranded DNA in acell, comprising a step of contacting a complex wherein a nucleic acidsequence-recognizing module that specifically binds to a targetnucleotide sequence in a given double stranded DNA and DNA glycosylasewith sufficiently low reactivity with a DNA having an unrelaxed doublehelix structure (unrelaxed DNA) are bonded, with said double strandedDNA, to convert one or more nucleotides in the targeted site to otherone or more nucleotides or delete one or more nucleotides, or insert oneor more nucleotides into said targeted site, without cleaving at leastone strand of said double stranded DNA in the targeted site.[2] The method of the above-mentioned [1], wherein the aforementionednucleic acid sequence-recognizing module is selected from the groupconsisting of a CRISPR-Cas system wherein at least one DNA cleavageability of Cas is inactivated, a zinc finger motif, a TAL effector and aPPR motif.[3] The method of the above-mentioned [1], wherein the aforementionednucleic acid sequence-recognizing module is a CRISPR-Cas system whereinat least one DNA cleavage ability of Cas is inactivated.[4] The method of the above-mentioned [1] or [2], wherein the doublestranded DNA is further contacted with a factor that changes a DNAdouble stranded structure.[5] The method of any of the above-mentioned [1]-[4], which uses two ormore kinds of nucleic acid sequence-recognizing modules eachspecifically binding to a different target nucleotide sequence.[6] The method of the above-mentioned [5], wherein the aforementioneddifferent target nucleotide sequences are present in different genes.[7] The method of any of the above-mentioned [1]-[6], wherein theaforementioned DNA glycosylase has cytosine-DNA glycosylase (CDG)activity or thymine-DNA glycosylase (TDG) activity.[8] The method of the above-mentioned [7], wherein the aforementionedDNA glycosylase having CDG activity or TDG activity is a mutant ofuracil-DNA glycosylase (UDG).[9] The method of any of the above-mentioned [1]-[8], further comprisingcontacting the double stranded DNA with an AP endonuclease havingbinding capacity to an abasic site but lacking nuclease activity.[10] The method of any of the above-mentioned [1]-[9], wherein theaforementioned DNA glycosylase has natively low reactivity with a DNAhaving an unrelaxed double helix structure.[11] The method of the above-mentioned [10], wherein the aforementionedDNA glycosylase is a mutant of uracil-DNA glycosylase (UDG) derived froma virus belonging to Poxviridae and having CDG activity or TDG activity.[12] The method of the above-mentioned [11], wherein the double strandedDNA is further contacted with A20 protein.[13] The method of any of the above-mentioned [1]-[9], wherein theaforementioned DNA glycosylase is a mutant having reduced reactivitywith a DNA having an unrelaxed double helix structure (unrelaxed DNA) ascompared to a wild-type one.[14] The method of any of the above-mentioned [1]-[9], wherein theaforementioned DNA glycosylase, and an element of the aforementionednucleic acid sequence-recognizing module which is directly bonded to theDNA glycosylase are respectively split into two fragments, the fragmentsof either of the DNA glycosylase and the element are respectively linkedto the fragments of the other to provide two partial complexes, and whenthe partial complexes are refolded with each other, the nucleic acidsequence-recognizing module is capable of specifically binding to thetarget nucleotide sequence and the specific bond enables the DNAglycosylase to exhibit enzyme activity.[15] The method of the above-mentioned [14], wherein the element of thenucleic acid sequence-recognizing module which is directly bonded to theaforementioned DNA glycosylase is a Cas protein wherein at least one ofthe DNA cleavage abilities is inactivated.[16] The method of the above-mentioned [14] or [15], wherein theaforementioned two partial complexes are provided as separate moleculecomplexes, and are refolded by association thereof in the cell.[17] The method of any of the above-mentioned [1]-[16], wherein thedouble stranded DNA is contacted with the complex by introducing anucleic acid encoding the complex into a cell having the double strandedDNA.[18] The method of the above-mentioned [17], wherein the aforementionedcell is a prokaryotic cell.[19] The method of the above-mentioned [17], wherein the aforementionedcell is a eukaryotic cell.[20] The method of the above-mentioned [17], wherein the aforementionedcell is a microbial cell.[21] The method of the above-mentioned [17], wherein the aforementionedcell is a plant cell.[22] The method of the above-mentioned [17], wherein the aforementionedcell is an insect cell.[23] The method of the above-mentioned [17], wherein the aforementionedcell is an animal cell.[24] The method of the above-mentioned [17], wherein the aforementionedcell is a vertebrate cell.[25] The method of the above-mentioned [17], wherein the aforementionedcell is a mammalian cell.[26] The method of any of the above-mentioned [18]-[25], wherein theaforementioned cell is a polyploid cell, and all of the targeted sitesin alleles on a homologous chromosome are modified.[27] A nucleic acid-modifying enzyme complex wherein a nucleic acidsequence-recognizing module that specifically binds to a targetnucleotide sequence in a given double stranded DNA and DNA glycosylasewith sufficiently low reactivity with a DNA having an unrelaxed doublehelix structure (unrelaxed DNA) are bonded, which converts one or morenucleotides in the targeted site to other one or more nucleotides ordeletes one or more nucleotides, or inserts one or more nucleotides intosaid targeted site, without cleaving at least one strand of said doublestranded DNA in the targeted site.[28] A nucleic acid encoding the nucleic acid-modifying enzyme complexof the above-mentioned [27].

Effect of the Invention

According to the genome editing technique of the present invention,since it does not accompany insertion of a foreign DNA or doublestranded DNA breaks, the technique is superior in safety, and has nosmall possibility of affording a solution to cases causing biological orlegal disputes on conventional methods as relating to generecombination. It is also possible to set an appropriate range ofmutation introduction up to the range of several hundred bases includingthe target sequence, and the technique can also be applied to topicalevolution induction by introduction of random mutation into a particularrestricted region, which has been almost impossible heretofore.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing of the mechanism of DNA sequence specifictargeting of uracil-DNA glycosylase mutant.

FIG. 2 is a schematic showing of a mechanism of the genetic modificationmethod of the present invention using DNA glycosylase and the CRISPR-Cassystem.

FIG. 3 shows the results of verification, by using a budding yeast, ofthe effect of the genetic modification method of the present inventioncomprising a CRISPR-Cas system and mutant uracil-DNA glycosylase derivedfrom budding yeast (having CDG activity and decreased reactivity withDNA having unrelaxed double helix structure) in combination.

FIG. 4 shows the results of verification, by using a budding yeast, ofthe effect of the genetic modification method of the present inventioncomprising a CRISPR-Cas system and budding yeast-derived mutanturacil-DNA glycosylase (having CDG activity or TDG activity anddecreased reactivity with DNA having unrelaxed double helix structure)in combination.

FIG. 5 shows the analysis results of mutated sites in thecanavanine-resistant colony obtained in FIG. 4.

FIG. 6 shows the results when an expression construct constructed suchthat budding yeast-derived mutant uracil-DNA glycosylase and dCas9 arebound via SH3 domain and a binding ligand thereof is introduced into abudding yeast together with two kinds of gRNA.

FIG. 7 shows that the efficiency of mutation induction by mutant UNG1(“Ung” in Figure means UNG1 derived from yeast, hereinafter the same) isimproved by coexpression of AP endonuclease (Ape1) mutants (E96Q,D210N).

FIG. 8 shows that cytotoxicity in a host yeast introduced with mutantUNG1 having CDG activity (N222D) or TDG activity (Y164A) can be avoidedby introducing a mutation (L304A) that decreases reactivity with a DNAhaving an unrelaxed double helix structure.

FIG. 9 shows survival rate and efficiency of mutation induction in ayeast introduced with a heterogenous mutant uracil-DNA glycosylasederived from Escherichia coli or vaccinia virus (EcUDG or vvUDG). vvUDGdid not cause cytotoxicity in a host yeast even without introduction ofa mutation (R187C) that decreases reactivity with a DNA having anunrelaxed double helix structure. The efficiency of mutation inductionby vvUDG was remarkably improved by coexpression of A20 protein.

FIG. 10 shows comparison of mutation introduction sites between mutantUNG1 derived from yeast and mutant UDG derived from vaccinia virus(vvUDG) in the target nucleotide sequences and in the vicinity thereof.

FIG. 11 shows that non-specific mutation by mutant UNG1 can be reducedby utilizing a split enzyme.

DESCRIPTION OF EMBODIMENTS

The present invention provides a method of modifying a nucleotide of atargeted site of a double stranded DNA by utilizing an error in baseexcision repair (BER) of cells, by excising a base from a nucleotide ina single strand region or in a region in which the double helixstructure is relaxed in the target nucleotide sequence of the doublestranded DNA and in the vicinity thereof, without cleaving at least onestrand of the double stranded DNA to be modified. The method ischaracterized in that it contains a step of contacting a complex whereinthe nucleic acid sequence-recognizing module that specifically binds tothe target nucleotide sequence in the double stranded DNA, and DNAglycosylase with sufficiently low reactivity with a DNA having anunrelaxed double helix structure are bonded with the double stranded DNAto convert the targeted site, i.e., the target nucleotide sequence andnucleotides in the vicinity thereof, to other nucleotides or deletesame, or insert one or more nucleotides into the targeted site.

In the present invention, the “modification” of a double stranded DNAmeans that a nucleotide (e.g., dC) on a DNA strand is converted to othernucleotide (e.g., dT, dA or dG), or deleted, or a nucleotide or anucleotide sequence is inserted between certain nucleotides on a DNAstrand. While the double stranded DNA to be modified is not particularlylimited, it is preferably a genomic DNA. The “targeted site” of a doublestranded DNA means the whole or partial “target nucleotide sequence”,which a nucleic acid sequence-recognizing module specifically recognizesand binds to, or the vicinity of the target nucleotide sequence (one orboth of 5′ upstream and 3′ downstream), and the range thereof can beappropriately adjusted between 1 base and several hundred basesaccording to the object.

In the present invention, the “nucleic acid sequence-recognizing module”means a molecule or molecule complex having an ability to specificallyrecognize and bind to a particular nucleotide sequence (i.e., targetnucleotide sequence) on a DNA strand. Binding of the nucleic acidsequence-recognizing module to a target nucleotide sequence enables aDNA glycosylase linked to the module to specifically act on a targetedsite of a double stranded DNA.

In the present invention “DNA glycosylase” means an enzyme thathydrolyzes N-glycosidic bond of DNA. DNA glycosylase originally plays arole of eliminating injured bases from DNA in BER. In the presentinvention, DNA glycosylase capable of acting on normal bases (i.e., dC,dT, dA and dG, or those after epigenetic modification) in DNA ispreferable. A mutant DNA glycosylase which natively does not react witha normal base or has low reactivity but which acquired reactivity or hasimproved reactivity with a normal base by mutation is encompassed in theDNA glycosylase in the present invention and can be preferably used. Anabasic site (apurinic/apyrimidic (AP) site) resulting from the baseexcision reaction by the enzyme is treated with an enzyme at thedownstream of the BER pathway, such as AP endonuclease, DNA polymerase,DNA ligase and the like.

With “sufficiently low reactivity with a DNA having an unrelaxed doublehelix structure” means that a base excision reaction in a region where aDNA having an unrelaxed double helix structure is formed is performedonly at a frequency sufficient to suppress cytotoxicity to a leveluninfluential on the survival of the cells. As used herein, a “DNAhaving an unrelaxed double helix structure” refers to having formed afirm double helix structure (namely, unrelaxed double-helical DNA (orsimply unrelaxed DNA)), and does not include the state of single strandDNA from completely dissociated pairing bases, and the state of doublestrands in an unwound and relaxed double helix structure forming basepairs (relaxed double-stranded DNA). Examples of the DNA glycosylasewith sufficiently low reactivity with a DNA having an unrelaxed doublehelix structure include DNA glycosylase with natively sufficiently lowreactivity with a DNA having an unrelaxed double helix structure, mutantDNA glycosylase introduced with a mutation to decrease reactivity with aDNA having an unrelaxed double helix structure as compared to wild-typeand the like. Furthermore, DNA glycosylase which is a split enzymedesigned to be split into two fragments, wherein respective fragmentsare bonded to either one of the nucleic acid sequence-recognizing modulesplit into two to form two complexes, when the both complexes arerefolded, the nucleic acid sequence-recognizing module can bespecifically bonded to the target nucleotide sequence, and, due to thespecific bond, the DNA glycosylase can catalize the base excisionreaction is also encompassed in the “DNA glycosylase with sufficientlylow reactivity with a DNA having an unrelaxed double helix structure” inthe present invention.

In the present invention, the “nucleic acid-modifying enzyme complex”means a molecule complex comprising a complex wherein theabove-mentioned nucleic acid sequence-recognizing module and a DNAglycosylase with sufficiently low reactivity with a DNA having anunrelaxed double helix structure are linked, which molecule complex isimparted with a particular nucleotide sequence recognition ability, andan activity to catalyze a base excision reaction of nucleic acid. The“complex” here encompasses not only one constituted of multiplemolecules, but also one having a nucleic acid sequence-recognizingmodule and DNA glycosylase in a single molecule, like a fusion protein.In addition, the “nucleic acid-modifying enzyme complex” in the presentinvention also encompasses a molecule complex in which nucleic acidsequence-recognizing module split into two fragments and eitherfragments of DNA glycosylase split into two fragments are linked to eachother to form two “partial complexes”, and the molecule complex acquiresa nucleotide sequence recognition ability and a base excision reactioncatalyst activity when the partial complexes are refolded with eachother.

While DNA glycosylase to be used in the present invention is notparticularly limited as long as it can hydrolyze N-glycosidic bond ofDNA to catalyze a reaction for releasing a base, DNA glycosylase capableof acting on normal bases (i.e., dC, dT, dA and dG, or those afterepigenetic modification, for example, 5-methylcytosine etc.) in DNA ispreferable to increase versatility as a genome editing technique.Examples of such enzyme include an enzyme having CDG activity tocatalyze a reaction for releasing cytosine, an enzyme having TDGactivity to catalyze a reaction for releasing thymine, an enzyme havingactivity to catalyze a reaction for releasing 5-methylcytosine (5-mCDGactivity) and the like. While DNA glycosylase natively having CDGactivity has not been reported to date, in almost all species, a mutantUDG having CDG activity or TDG activity that recognizes cytidine orthymidine as a substrate can be obtained by introducing a mutation intoa particular amino acid residue of UNG known as the major UDGresponsible for the removal of uracil incorporated in DNA (in the caseof eucaryote, it has two splice variants of mitochondrial localizationUNG1 and nuclear localization UNG2, which have a common amino acidsequence except N-terminal sequence containing each oraganellelocalization signal) (see FIG. 1, upper panel). More specifically, forexample, in the case of UNG1 derived from yeast (SEQ ID NO: 2; GenBankaccession No. NP_013691), CDG activity can be imparted by substitutingthe 222nd asparagine from the N-terminal with aspartic acid (the mutantis also referred to as N222D), and TDG activity can be imparted bysubstituting the 164th tyrosine with alanine (the mutant is alsoreferred to as Y164A) (Kavli B. et al., EMBO J. (1996) 15(13): 3442-7).The present inventors have newly found that TDG activity higher thanthat of Y164A mutant can be imparted by substituting the 164th tyrosinewith glycine (the mutant is also referred to as Y164G). Furthermore,since cytosine is obtained by substituting the carbonyl group at the4-position of uracil with an amino group and thymine is equivalent touracil in which the 5-position is methylated, DNA glycosylase having anactivity to release 5-methylcytosine, derived from methylation of the5-position of cytosine, from DNA (5-mCDG activity) can be obtained byintroducing double mutation into the 222nd asparagine residue and the164th tyrosine residue (e.g., N222D/Y164A or N222D/Y164G). Since baseexcision of 5-methylcytosine can change the methylation state of thegenomic DNA, the genome editing technique of the present invention alsoenables epigenome editing to change epigenetic modification.

When UNG2 is used, mutant UNG having CDG activity, TDG activity or5-mCDG activity can be obtained by introducing similar mutation into theamino acid residue corresponding to the above-mentioned mutated site.

In the mutant UNG, the above-mentioned site may be substituted with anamino acid other than the above-mentioned amino acid, or the mutationmay be introduced into a site other than the above-mentioned site, aslong as it can act on a normal base. Whether the mutant UNG can act on anormal base can be confirmed by the method described in, for example,Kavli B. et al., EMBO J. (1996) 15(13): 3442-7.

While the derivation of UNG is not particularly limited, for example,ung derived Escherichia coli (Varshney, U. et al. (1988) J. Biol. Chem.,263, 7776-7784), UNG1 or UNG2 derived from yeast, mammal (e.g., human,mouse, swine, bovine, horse, monkey etc.) and the like, or UDG derivedfrom virus (e.g., Poxviridae (vaccinia virus etc.), Herpesviridae andthe like) can be used. For example, UniprotKB Nos. P13051-2 and P13051-1can be referred to for the amino acid sequences of human UNG1 and UNG2,respectively. In addition, UniprotKB No. P12295 can be referred to forthe amino acid sequence of Escherichia coli (K-12 strain) ung, andUniprotKB No. P20536 can be referred to for the amino acid sequence ofvaccinia virus (Copenhagen Strain) UDG. The amino acid sequence of UNGis highly preserved among species, and the corresponding site formutation can be identified by aligning the amino acid sequence of UNG1or UNG2 derived from a desired organism with that of the above-mentionedyeast UNG1. For example, in the case of human UNG1, the amino acidcorresponding to N222 of yeast UNG1 is the 204th asparagine (N204), andthe amino acid corresponding to Y164 is the 147th tyrosine (Y147). Inthe case of Escherichia coli ung, the amino acid corresponding to N222of yeast UNG1 is the 123rd asparagine (N123), and the amino acidcorresponding to Y164 is the 66th tyrosine (Y66). In the case of UDGderived from Poxviridae virus such as vaccinia virus, smallpoxvirus,monkeypoxvirus, fowlpox virus, swinepox virus, rabbit fibroma virus, theamino acid corresponding to N222 of yeast UNG1 is the 120th asparagine(N120), and the amino acid corresponding to Y164 is the 70th tyrosine(Y70).

While UNG can remove uracil from single stranded DNA and double strandedDNA, it has higher affinity for single stranded DNA. This tendency isalso found in the above-mentioned mutant UNG conferred with CDG activityand TDG activity. However, since cytosine and thymine exist everywherein the genomic DNA, mutant UNG having CDG activity or TDG activity acts,unlike wild-type UNG that exclusively uses, as a substrate, uracil whichis rarely introduced into genomic DNA by an error on replication ordeamination of cytosine, on any site in the nucleotide sequence targetedby a nucleic acid sequence-recognizing module and double stranded DNA inthe vicinity thereof to remove cytosine or thymine, thus causing strongcytotoxicity. Therefore, DNA glycosylase to be used in the presentinvention is required to show sufficiently low reactivity with a DNAhaving an unrelaxed double helix structure. When mutant UNG is used asDNA glycosylase, the cytotoxicity by the enzyme can be avoided byfurther introducing a mutation that decreases reactivity with a DNAhaving an unrelaxed double helix structure to make a base excisionreaction by mutant UNG having CDG activity or TDG activity moreselective to the region of a relaxed double-stranded or single strandedDNA (see FIG. 1, middle panel). Specifically, for example, in the caseof UNG, a mutation that decreases the reactivity with U-A 25 mer doublestranded DNA to not more than 1/20, more preferably not more than 1/50,further preferably not more than 1/100, in vitro, that of the wild-typecan be mentioned. However, as long as the reactivity with a DNA havingan unrelaxed double helix structure is decreased to a level free fromlethal cytotoxicity in vivo, it is not limited by the reactivity invitro. Whether the DNA glycosylase to be used has “sufficiently lowreactivity with a DNA having an unrelaxed double helix structure” can beconfirmed, for example, by inserting the DNA glycosylase into theconstruct described in FIG. 2, introducing the construct together withthe guide RNA into the object cell by the method described in Example 1,culturing the obtained transformant, and verifying the survivabilitythereof. Alternatively, a candidate of DNA glycosylase with“sufficiently low reactivity with a DNA having an unrelaxed double helixstructure” can also be screened for by evaluating, as one of thecriteria, the reactivity with a double stranded DNA oligomer in vitro bythe method described in Chen, C. Y. et al. DNA Repair (Amst) (2005)4(7): 793-805.

As a DNA glycosylase fulfilling the above-mentioned conditions in thecase of, for example, UNG1 derived from yeast (SEQ ID NO: 2), a mutantin which the 304th leucine from the N-terminal is substituted withalanine can be mentioned (the mutant is also referred to as L304A)(Slupphaug, G. et al. Nature (1996) 384(6604): 87-92). Alternatively, amutant in which the 308th arginine is substituted with glutamic acid orcysteine (the mutants are also referred to as R308E, R308C,respectively) also shows remarkably decreased reactivity with a DNAhaving an unrelaxed double helix structure (Chen, C. Y. et al. DNARepair (Amst) (2005) 4(7): 793-805). When a different species derivedfrom UNG is used, the corresponding site for mutation can be identifiedby aligning the amino acid sequence of UNG1 or UNG2 derived from adesired organism with that of the above-mentioned yeast UNG1. Forexample, in the case of human UNG1, the amino acid corresponding to L304of yeast UNG1 is the 272nd leucine (L272), and the amino acidcorresponding to R308 is the 276th arginine (R276). In the case ofEscherichia coli ung, the amino acid corresponding to L304 of yeast UNG1is the 191st leucine (L191), and the amino acid corresponding to R308 isthe 195th arginine (R195). In the case of vaccinia virus UDG, the aminoacid corresponding to R308 of yeast UNG1 is the 187th arginine (R187).

The substrate specificity of UNG1(ung) to yeast (Saccharomycescerevisiae), Escherichia coli, human and Poxviridae virus, and the siteof mutation that changes the reactivity with a DNA having an unrelaxeddouble helix structure are shown in Table 1. Mutant amino acid isindicated with one letter, and the number shows the position of mutantamino acid when N-terminal amino acid is 1.

TABLE 1 Escherichia pox change of mutant form and yeast coli human virusphenotype Y164 Y66 Y147 Y70 recognizes thymine by mutation to A, G N222N123 N204 N120 recognizes cytosine by mutation to D L304 L191 L272 —reactivity with DNA having unrelaxed double helix structure decreases bymutation to A R308 R195 R276 R187^(a)) reactivity with DNA havingunrelaxed double helix structure decreases by mutation to E, C ^(a))Invaccinia virus UDG etc., wild-type itself is considered to have nativelylow reactivity with DNA having unrelaxed double helix structure.

From the above, preferable examples of the “DNA glycosylase withsufficiently low reactivity with a DNA having an unrelaxed double helixstructure” to be used in the present invention include N222D/L304Adouble mutant, N222D/R308E double mutant, N222D/R308C double mutant,Y164A/L304A double mutant, Y164A/R308E double mutant, Y164A/R308C doublemutant, Y164G/L304A double mutant, Y164G/R308E double mutant,Y164G/R308C double mutant, N222D/Y164A/L304A triple mutant,N222D/Y164A/R308E triple mutant, N222D/Y164A/R308C triple mutant,N222D/Y164G/L304A triple mutant, N222D/Y164G/R308E triple mutant,N222D/Y164G/R308C triple mutant and the like of yeast UNG1. Whendifferent UNG is used instead of yeast UNG1, a mutant having an aminoacid corresponding to each of the above-mentioned mutants, which isintroduced with a similar mutation, may be used.

Alternatively, as a “DNA glycosylase with sufficiently low reactivitywith a DNA having an unrelaxed double helix structure”, DNA glycosylasewith natively low reactivity with a DNA having an unrelaxed double helixstructure, and high selectivity to relaxed double stranded or singlestranded DNA can also be used. Examples of such DNA glycosylase includeSMUG1 having UDG activity (Single strand-selective MonofunctionalUracil-DNA Glycosylase). While a mutation conferring CDG activity or TDGactivity to SMUG1 is not known, it has been reported that the SMUG1 isimportant for removal of uracil resulting from deamination of cytosine(Nilsen, H. et al. EMBO J. (2001) 20: 4278-4286). The present inventorshave already developed a method for specifically introducing a mutationinto the targeted nucleotide sequence and the vicinity thereof, bycombining cytidine deaminase capable of converting cytosine to uracil,and a nucleic acid sequence-recognizing module similar to that in thepresent invention (WO 2015/133554). By combining with the technique,cytosine can be artificially converted to uracil in the targeted site ingenomic DNA, after which SMUG1 can be further reacted to release theuracil from DNA. While the derivation of SMUG1 is not particularlylimited, for example, SMUG1 derived from Escherichia coli, yeast, mammal(e.g., human, mouse, swine, bovine, horse, monkey etc.) and the like canbe used. For example, UniprotKB No. Q53HC7-1 and Q53HV7-2 can bereferred to for the two amino acid sequences of the two isoforms ofhuman SMUG1. In addition, while the derivation of cytidine deaminase isnot particularly limited, for example, Petromyzon marinus-derived PmCDA1(Petromyzon marinus cytosine deaminase 1), or AID (Activation-inducedcytidine deaminase; AICDA) derived from mammal (e.g., human, swine,bovine, horse, monkey etc.) can be used. For example, GenBank accessionNo. EF094822 and AB015149 can be referred to for the base sequence andthe amino acid sequence of PmCDA1 cDNA, and GenBank accession No. NM020661 and NP 065712 can be referred to for the base sequence and theamino acid sequence of human AID cDNA.

In another preferable embodiment, as the DNA glycosylase with nativelylow reactivity with a DNA having an unrelaxed double helix structure,UDG derived from virus belonging to Poxviridae such as vaccinia virusand the like can be mentioned. As shown in the below-mentioned Examples,vaccinia virus-derived UDG (vvUDG) is considered to be sufficiently lowin the reactivity with a DNA having an unrelaxed double helix structureto a level free from toxicity in a host cell, because it shows growthequivalent to that in yeast UNG1 and Escherichia coli ung, introducedwith a mutation that decreases reactivity with a DNA having an unrelaxeddouble helix structure (e.g., R187C), on a nonselective medium, evenwhen such mutation is not introduced. When UDG derived from Poxviridaevirus such as vvUDG and the like is used as a DNA glycosylase, amutation (e.g., R187C) corresponding to the mutation that decreasesreactivity with a DNA having an unrelaxed double helix structure inyeast UNG1 can be further introduced. However, when such mutationdecreases the CDG activity (N120D) and TDG activity (Y70A, Y70G) of UDG,it is desirably avoided. From the above, preferable examples of UDGderived from Poxviridae virus such as vvUDG to be used in the presentinvention include N120D mutant, Y70G or Y70A mutant, N120D/Y70G doublemutant or N120D/Y70A double mutant and the like.

When UDG derived from Poxviridae virus such as mutant vvUDG and the likeis used as the DNA glycosylase, it is preferable to contact A20 protein,which interacts with UDG to form a heterodimer that functions as aprocessivity factor of viral DNA polymerase, with double stranded DNAtogether with UDG. Since combined use with A20 protein increases CDGactivity or TDG activity of mutant UDG, for example, the efficiency ofmutation induction into thymine, of mutant vvUDG having low TDG activity(Y70G) as compared to CDG activity (N120D) can be improved by thecombined use with A20 protein. While the derivation of A20 is notparticularly limited, for example, A20 derived from virus belonging toPoxviridae such as vaccinia virus, smallpoxvirus, monkeypoxvirus,fowlpox virus, swinepox virus, rabbit fibroma virus can be used. Forexample, UniprotKB No. P20995 can be referred for the amino acidsequence of vaccinia virus (Copenhagen Strain) A20.

In yet another preferable embodiment, a split enzyme designed such thatnucleic acid sequence-recognizing module and DNA glycosylase are eachsplit into two fragments, either fragments are linked to each other toform two partial complexes, these complexes are associated toreconstitute a functional nucleic acid sequence-recognizing module, andthe module is bonded to the target nucleotide sequence to reconstitute afunctional DNA glycosylase can be used as a DNA glycosylase having lowreactivity with a DNA having an unrelaxed double helix structure. In thesplit enzyme, since the enzyme activity is exhibited only when it isbonded to the target nucleotide sequence, even when the DNA glycosylaseitself to be reconstituted does not have a reduced reactivity with a DNAhaving an unrelaxed double helix structure, it consequently actsselectively on the single stranded DNA or relaxed double stranded DNApart in the target nucleotide sequence and the vicinity thereof. Forexample, nucleic acid sequence-recognizing module and DNA glycosylaseare each split into N-terminal side fragments and C-terminal sidefragments, for example, N-terminal side fragments are linked to eachother to give a partial complex, C-terminal side fragments are linked toeach other to give a partial complex (or N-terminal side fragments ofnucleic acid sequence-recognizing module and C-terminal side fragmentsof DNA glycosylase are linked to give a partial complex, and N-terminalside fragments of DNA glycosylase and C-terminal side fragments ofnucleic acid sequence-recognizing module are linked to give a partialcomplex), and they are associated, whereby functional nucleic acidsequence-recognizing module and functional DNA glycosylase can bereconstituted. The combination of the fragments to be linked is notparticularly limited as long as a complex of the functional nucleic acidsequence-recognizing module and the functional DNA glycosylase isreconstituted when the two partial complexes are associated. The twopartial complexes may be provided as separate molecules, or may beprovided as a single fusion protein by linking them directly or via asuitable linker. The split site in DNA glycosylase is not particularlylimited as long as two split fragments can be reconstituted asfunctional DNA glycosylase, and DNA glycosylase may be split at one siteto provide N-terminal side fragment and C-terminal side fragment, or notless than 3 fragments obtained by splitting at two or more sites may beappropriately linked to give two fragments. The three-dimensionalstructures of various UDG proteins are known, and those of ordinaryskill in the art can appropriately select the split sites based on suchinformation. For example, yeast UNG1 (SEQ ID NO: 2) can be split betweenthe 258th amino acid and 259th amino acid from the N-terminal to giveN-terminal side fragment (1-258) and C-terminal side fragment (259-359).

As mentioned above, in the base excision repair (BER) mechanism, when abase is excised by DNA glycosylase, AP endonuclease puts a nick in theabasic site (AP site), and exonuclease completely excises the AP site.When the AP site is excised, DNA polymerase produces a new base by usingthe base of the opposing strand as a template, and DNA ligase finallyseals the nick to complete the repair. Mutant AP endonuclease that haslost the enzyme activity but maintains the binding capacity to the APsite is known to competitively inhibit BER. Therefore, when the mutantAP endonuclease is contacted with double stranded DNA together with DNAglycosylase, the repair of the AP site by endogenous BER mechanism inthe host cell is inhibited, and the frequency of repair errors, namely,efficiency of mutation induction, is improved. For example, theefficiency of mutation induction into thymine in mutant yeast UNG1having lower TDG activity (Y164G) as compared to CDG activity (N222D)can be improved by using mutant AP endonuclease in combination. Whilethe derivation of AP endonuclease is not particularly limited, forexample, AP endonuclease derived from Escherichia coli, yeast, mammal(e.g., human, mouse, swine, bovine, horse, monkey etc.) and the like canbe used. For example, UniprotKB No. P27695 can be referred to for theamino acid sequence of human Ape1. Examples of the mutant APendonuclease that has lost the enzyme activity but maintains the bindingcapacity to the AP site include proteins having mutated activity siteand mutated Mg (cofactor)-binding site. For example, E96Q, Y171A, Y171F,Y171H, D210N, D210A, N212A and the like can be mentioned for human Ape1.

A target nucleotide sequence in a double stranded DNA to be recognizedby the nucleic acid sequence-recognizing module in the nucleicacid-modifying enzyme complex of the present invention is notparticularly limited as long as the module specifically binds to, andmay be any sequence in the double stranded DNA. The length of the targetnucleotide sequence only needs to be sufficient for specific binding ofthe nucleic acid sequence-recognizing module. For example, when mutationis introduced into a particular site in the genomic DNA of a mammal, itis not less than 12 nucleotides, preferably not less than 15nucleotides, more preferably not less than 17 nucleotides, according tothe genome size thereof. While the upper limit of the length is notparticularly limited, it is preferably not more than 25 nucleotides,more preferably not more than 22 nucleotides.

As the nucleic acid sequence-recognizing module in the nucleicacid-modifying enzyme complex of the present invention, CRISPR-Cassystem wherein at least one DNA cleavage ability of Cas is inactivated(CRISPR-mutant Cas), zinc finger motif, TAL effector and PPR motif andthe like, as well as a fragment containing a DNA binding domain of aprotein that specifically binds to DNA, such as restriction enzyme,transcription factor, RNA polymerase and the like, and free of a DNAdouble strand cleavage ability and the like can be used, but the moduleis not limited thereto. Preferably, CRISPR-mutant Cas, zinc fingermotif, TAL effector, PPR motif and the like can be mentioned.

A zinc finger motif is constituted by linkage of 3-6 different Cys2His2type zinc finger units (1 finger recognizes about 3 bases), and canrecognize a target nucleotide sequence of 9-18 bases. A zinc fingermotif can be produced by a known method such as Modular assembly method(Nat Biotechnol (2002) 20: 135-141), OPEN method (Mol Cell (2008) 31:294-301), CoDA method (Nat Methods (2011) 8: 67-69), Escherichia colione-hybrid method (Nat Biotechnol (2008) 26:695-701) and the like. Theabove-mentioned patent document 1 can be referred to as for the detailof the zinc finger motif production.

A TAL effector has a module repeat structure with about 34 amino acidsas a unit, and the 12th and 13th amino acid residues (called RVD) of onemodule determine the binding stability and base specificity. Since eachmodule is highly independent, TAL effector specific to a targetnucleotide sequence can be produced by simply connecting the module. ForTAL effector, a production method utilizing an open resource (REALmethod (Curr Protoc Mol Biol (2012) Chapter 12: Unit 12.15), FLASHmethod (Nat Biotechnol (2012) 30: 460-465), and Golden Gate method(Nucleic Acids Res (2011) 39: e82) etc.) have been established, and aTAL effector for a target nucleotide sequence can be designedcomparatively conveniently. The above-mentioned patent document 2 can bereferred to as for the detail of the production of TAL effector.

PPR motif is constituted such that a particular nucleotide sequence isrecognized by a continuation of PPR motifs each consisting of 35 aminoacids and recognizing one nucleic acid base, and recognizes a targetbase only by 1, 4 and ii(-2) amino acids of each motif. Motifconstitution has no dependency, and is free of interference of motifs onboth sides. Therefore, like TAL effector, a PPR protein specific to thetarget nucleotide sequence can be produced by simply connecting PPRmotifs. The above-mentioned patent document 4 can be referred to as forthe detail of the production of PPR motif.

When a fragment of restriction enzyme, transcription factor, RNApolymerase and the like is used, since the DNA binding domains of theseproteins are well known, a fragment containing the domain and free of aDNA double strand cleavage ability can be easily designed andconstructed.

As mentioned above, DNA glycosylase used for the nucleic acid-modifyingenzyme complex of the present invention is preferably mutant UDGconferred with CDG activity or TDG activity, more preferably UNG, and itneeds to be sufficiently low in the reactivity with a DNA having anunrelaxed double helix structure so that CDG activity or TDG activitywill not act on anywhere in the targeted site (see FIG. 1, middlepanel). Therefore, the targeted site is desirably in the state of singlestranded DNA, or relaxed DNA structure resulting from unwinding of atleast firm double helix structure, which enables the DNA glycosylase toact efficiently when brought into contact with DNA glycosylase. WhenCRISPR-Cas system is used as the nucleic acid sequence-recognizingmodule, guide RNA complementary to the target nucleotide sequencerecognizes the sequence of the object double stranded DNA andspecifically forms a hybrid with the target nucleotide sequence, wherebythe targeted site becomes the state of single strand or unwound doublehelix structure (relaxed double stranded) state. Therefore, DNAglycosylase with sufficiently low reactivity with a DNA having anunrelaxed double helix structure selectively acts on cytosine or thyminein the targeted site and can excise the base (see FIG. 1, lower panel).On the other hand, when zinc finger motif, TAL effector, PPR motif andthe like are used as the nucleic acid sequence-recognizing module, sincethe module itself does not have a function to change the structure ofdouble stranded DNA (cause distortion of double helix structure), it isdesirable to contact the nucleic acid-modifying enzyme complex of thepresent invention in combination with a factor (e.g., gyrase,topoisomerase, helicase etc.), which changes the structure of the objectdouble stranded DNA, with the double stranded DNA.

Any of the above-mentioned nucleic acid sequence-recognizing module canbe provided as a fusion protein with the above-mentioned DNAglycosylase, or a protein binding domain such as SH3 domain, PDZ domain,GK domain, GB domain and the like and a binding partner thereof may befused with a nucleic acid sequence-recognizing module and a DNAglycosylase, respectively, and provided as a protein complex via aninteraction of the domain and a binding partner thereof. Alternatively,a nucleic acid sequence-recognizing module and a DNA glycosylase may beeach fused with intein, and they can be linked by ligation after proteinsynthesis.

The nucleic acid-modifying enzyme complex of the present inventioncontaining a complex (including fusion protein) wherein a nucleic acidsequence-recognizing module and DNA glycosylase are bonded is contactedwith a double stranded DNA (e.g., genomic DNA) by introducing thecomplex or a nucleic acid encoding the complex into a cell having theobject double stranded DNA. In consideration of the introduction andexpression efficiency, it is desirable to introduce the nucleicacid-modifying enzyme complex into the cell in the form of a nucleicacid encoding the complex, rather than the complex itself, and allow forexpression of the complex in the cell. Also when mutant AP endonuclease,A20 protein and the like are used in combination, it is desirable tointroduce them into the cell in the form of a nucleic acid encodingthem, and allow for expression of the complex in the cell.

Therefore, the nucleic acid sequence-recognizing module and the DNAglycosylase are preferably prepared as a nucleic acid encoding a fusionprotein thereof, or in a form capable of forming a complex in a hostcell after translation into a protein by utilizing a binding domain,intein and the like, or as a nucleic acid encoding each of them. Thenucleic acid here may be a DNA or an RNA. When it is a DNA, it ispreferably a double stranded DNA, and provided in the form of anexpression vector disposed under regulation of a functional promoter ina host cell. When it is an RNA, it is preferably a single stranded RNA.

When nucleic acid sequence-recognizing module and DNA glycosylase areeach split into two fragments, the fragments of either of them arerespectively linked to the fragments of the other to provide two partialcomplexes, for example, a DNA encoding the N-terminal side fragment anda DNA encoding the C-terminal side fragment of the nucleic acidsequence-recognizing module are respectively prepared by the PCR methodusing suitable primers; and a DNA encoding the N-terminal side fragmentand a DNA encoding the C-terminal side fragment of DNA glycosylase areprepared in the same manner and, for example, the DNAs encoding theN-terminal side fragments, and the DNAs encoding the C-terminal sidefragments are linked to each other by a conventional method, whereby aDNA encoding the two partial complexes can be produced. Alternatively, aDNA encoding the N-terminal side fragment of the nucleic acidsequence-recognizing module and a DNA encoding the C-terminal sidefragment of the DNA glycosylase are linked; and a DNA encoding theN-terminal side fragment of the DNA glycosylase and a DNA encoding theC-terminal side fragment of the nucleic acid sequence-recognizing moduleare linked, whereby a DNA encoding the two partial complexes can also beproduced. The combination of the fragments to be linked is notparticularly limited as long as a complex of the functional nucleic acidsequence-recognizing module and the functional DNA glycosylase isreconstituted when the two partial complexes are associated. The twopartial complexes are not only expressed as separate molecules, but mayalso be expressed as a single fusion protein by linking nucleic acidsencoding them directly or via a suitable linker, which protein forms acomplex of the functional nucleic acid sequence-recognizing module andthe functional DNA glycosylase by intramolecular association.

Since the complex of the present invention wherein a nucleic acidsequence-recognizing module and a DNA glycosylase are bonded does notaccompany double stranded DNA breaks (DSB), genome editing with lowtoxicity is possible, and the genetic modification method of the presentinvention can be applied to a wide range of biological materials.Therefore, the cells to be introduced with nucleic acid encoding nucleicacid sequence-recognizing module and/or DNA glycosylase can encompasscells of any species, from bacterium of Escherichia coli and the likewhich are prokaryotes, cells of microorganism such as yeast and the likewhich are lower eucaryotes, to cells of vertebrata including mammalssuch as human and the like, and cells of higher eukaryote such asinsect, plant and the like.

A DNA encoding a nucleic acid sequence-recognizing module such as zincfinger motif, TAL effector, PPR motif and the like can be obtained byany method mentioned above for each module. A DNA encoding asequence-recognizing module of restriction enzyme, transcription factor,RNA polymerase and the like can be cloned by, for example, synthesizingan oligoDNA primer covering a region encoding a desired part of theprotein (part containing DNA binding domain) based on the cDNA sequenceinformation thereof, and amplifying by the RT-PCR method using, as atemplate, the total RNA or mRNA fraction prepared from theprotein-producing cells.

A DNA encoding DNA glycosylase can also be cloned similarly bysynthesizing an oligoDNA primer based on the cDNA sequence informationthereof, and amplifying by the RT-PCR method using, as a template, thetotal RNA or mRNA fraction prepared from the enzyme-producing cells. Forexample, a DNA encoding UNG1 of yeast can be cloned by designingsuitable primers for the upstream and downstream of CDS based on thecDNA sequence (accession No. NM_001182379) registered in the NCBIdatabase, and cloning from yeast-derived mRNA by the RT-PCR method.

A nucleic acid encoding DNA glycosylase with sufficiently low reactivitywith a DNA having an unrelaxed double helix structure can be obtained bya site specific mutagenesis method known per se by using the obtainedcDNA as a template, and introducing a mutation imparting CDG activity,TDG activity or 5-mCDG activity, and a mutation that decreasesreactivity with a DNA having an unrelaxed double helix structure. WhenDNA glycosylase with natively sufficiently low reactivity with a DNAhaving an unrelaxed double helix structure, such as vvUDG and the like,only a mutation imparting CDG activity, TDG activity or 5-mCDG activitycan be introduced.

The cloned DNA may be directly, or after digestion with a restrictionenzyme when desired, or after addition of a suitable linker (e.g., GSlinker, GGGAR linker etc.), spacer (e.g., FLAG sequence etc.) and/or anuclear localization signal (NLS) (each organelle localization signalwhen the object double stranded DNA is mitochondria or chloroplast DNA),ligated with a DNA encoding a nucleic acid sequence-recognizing moduleto prepare a DNA encoding a fusion protein. Since UNG1 and UNG2 eachhave a mitochondria localization signal and a nuclear localizationsignal on the N-terminal, they may also be utilized as they are.Alternatively, for example, when UNG1 is used for nucleotidemodification targeting nuclear genomic DNA, it is possible to remove themitochondria localization signal and separately link a nuclearlocalization signal.

Alternatively, a DNA encoding a nucleic acid sequence-recognizingmodule, and a DNA encoding a DNA glycosylase may be each fused with aDNA encoding a binding domain or a binding partner thereof, or both DNAsmay be fused with a DNA encoding a separation intein, whereby thenucleic acid sequence-recognizing conversion module and the DNAglycosylase are translated in a host cell to form a complex. In thesecases, a linker and/or a nuclear localization signal can be linked to asuitable position of one of or both DNAs when desired.

A DNA encoding a nucleic acid sequence-recognizing module and a DNAencoding a DNA glycosylase can be obtained by chemically synthesizingthe DNA strand, or by connecting synthesized partly overlapping oligoDNAshort strands by utilizing the PCR method and the Gibson Assembly methodto construct a DNA encoding the full length thereof. The advantage ofconstructing a full-length DNA by chemical synthesis or a combination ofPCR method or Gibson Assembly method is that the codon to be used can bedesigned in CDS full-length according to the host into which the DNA isintroduced. In the expression of a heterologous DNA, the proteinexpression level is expected to increase by converting the DNA sequencethereof to a codon highly frequently used in the host organism. As thedata of codon use frequency in host to be used, for example, the geneticcode use frequency database (www.kazusa.or.jp/codon/index.html)disclosed in the home page of Kazusa DNA Research Institute can be used,or documents showing the codon use frequency in each host may bereferred to. By reference to the obtained data and the DNA sequence tobe introduced, codons showing low use frequency in the host from amongthose used for the DNA sequence may be converted to a codon coding thesame amino acid and showing high use frequency.

An expression vector containing a DNA encoding a nucleic acidsequence-recognizing module and/or a DNA glycosylase can be produced,for example, by linking the DNA to the downstream of a promoter in asuitable expression vector.

As the expression vector, Escherichia coli-derived plasmids (e.g.,pBR322, pBR325, pUC12, pUC13); Bacillus subtilis-derived plasmids (e.g.,pUB110, pTP5, pC194); yeast-derived plasmids (e.g., pSH19, pSH15);insect cell expression plasmids (e.g., pFast-Bac); animal cellexpression plasmids (e.g., pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo);bacteriophages such as Aphage and the like; insect virus vectors such asbaculovirus and the like (e.g., BmNPV, AcNPV); animal virus vectors suchas retrovirus, vaccinia virus, adenovirus and the like, and the like areused.

As the promoter, any promoter appropriate for a host to be used for geneexpression can be used. In a conventional method using DSB, since thesurvival rate of the host cell sometimes decreases markedly due to thetoxicity, it is desirable to increase the number of cells by the startof the induction by using an inductive promoter. However, sincesufficient cell proliferation can also be afforded by expressing thenucleic acid-modifying enzyme complex of the present invention, aconstitution promoter can also be used without limitation.

For example, when the host is an animal cell, SRa promoter, SV40promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Roussarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR,HSV-TK (simple herpes virus thymidine kinase) promoter and the like areused. Of these, CMV promoter, SRa promoter and the like are preferable.

When the host is Escherichia coli, trp promoter, lac promoter, recApromoter, λP_(L) promoter, 1pp promoter, T7 promoter and the like arepreferable.

When the host is genus Bacillus, SPO1 promoter, SPO2 promoter, penPpromoter and the like are preferable.

When the host is a yeast, Gal/10 promoter, PHO5 promoter, PGK promoter,GAP promoter, ADH promoter and the like are preferable.

When the host is an insect cell, polyhedrin promoter, P10 promoter andthe like are preferable.

When the host is a plant cell, CaMV35S promoter, CaMV19S promoter, NOSpromoter and the like are preferable.

As the expression vector, besides those mentioned above, one containingenhancer, splicing signal, terminator, polyA addition signal, aselection marker such as drug resistance gene, auxotrophic complementarygene and the like, replication origin and the like on demand can beused.

An RNA encoding a nucleic acid sequence-recognizing module and/or a DNAglycosylase can be prepared by, for example, transcription to mRNA in avitro transcription system known per se by using a vector encoding DNAencoding the above-mentioned nucleic acid sequence-recognizing moduleand/or a DNA glycosylase as a template.

A complex of a nucleic acid sequence-recognizing module and a DNAglycosylase can be intracellularly expressed by introducing anexpression vector containing a DNA encoding a nucleic acidsequence-recognizing module and/or a DNA glycosylase into a host cell,and culturing the host cell.

As the host, genus Escherichia, genus Bacillus, yeast, insect cell,insect, animal cell and the like are used.

As the genus Escherichia, Escherichia coli K12⋅DH1 [Proc. Natl. Acad.Sci. USA, 60, 160 (1968)], Escherichia coli JM103 [Nucleic AcidsResearch, 9, 309 (1981)], Escherichia coli JA221 [Journal of MolecularBiology, 120, 517 (1978)], Escherichia coli HB101 [Journal of MolecularBiology, 41, 459 (1969)], Escherichia coli C600 [Genetics, 39, 440(1954)] and the like are used.

As the genus Bacillus, Bacillus subtilis MI114 [Gene, 24, 255 (1983)],Bacillus subtilis 207-21 [Journal of Biochemistry, 95, 87 (1984)] andthe like are used.

As the yeast, Saccharomyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-5D,20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastorisKM71 and the like are used.

As the insect cell when the virus is AcNPV, cells of cabbage armywormlarva-derived established line (Spodoptera frugiperda cell; Sf cell),MG1 cells derived from the mid-intestine of Trichoplusia ni, High Five™cells derived from an egg of Trichoplusia ni, Mamestra brassicae-derivedcells, Estigmena acrea-derived cells and the like are used. When thevirus is BmNPV, cells of Bombyx mori-derived established line (Bombyxmori N cell; BmN cell) and the like are used as insect cells. As the Sfcell, for example, Sf9 cell (ATCC CRL1711), Sf21 cell [all above, InVivo, 13, 213-217 (1977)] and the like are used.

As the insect, for example, larva of Bombyx mori, Drosophila, cricketand the like are used [Nature, 315, 592 (1985)].

As the animal cell, cell lines such as monkey COS-7 cell, monkey Verocell, Chinese hamster ovary (CHO) cell, dhfr gene-deficient CHO cell,mouse L cell, mouse AtT-20 cell, mouse myeloma cell, rat GH3 cell, humanFL cell and the like, pluripotent stem cells such as iPS cell, ES celland the like of human and other mammals, and primary cultured cellsprepared from various tissues are used. Furthermore, zebrafish embryo,Xenopus oocyte and the like can also be used.

As the plant cell, suspend cultured cells, callus, protoplast, leafsegment, root segment and the like prepared from various plants (e.g.,grain such as rice, wheat, corn and the like, product crops such astomato, cucumber, egg plant and the like, garden plants such ascarnation, Eustoma russellianum and the like, experiment plants such astobacco, Arabidopsis thaliana and the like, and the like) are used.

All the above-mentioned host cells may be haploid (monoploid), orpolyploid (e.g., diploid, triploid, tetraploid and the like). In theconventional mutation introduction methods, mutation is, in principle,introduced into only one homologous chromosome to produce a hetero genetype. Therefore, desired phenotype is not expressed unless dominantmutation occurs, and homozygousness inconveniently requires labor andtime. In contrast, according to the present invention, since mutationcan be introduced into any allele on the homologous chromosome in thegenome, desired phenotype can be expressed in a single generation evenin the case of recessive mutation, which is extremely useful since theproblem of the conventional method can be solved.

An expression vector can be introduced by a known method (e.g., lysozymemethod, competent method, PEG method, CaCl₂ coprecipitation method,electroporation method, the microinjection method, the particle gunmethod, lipofection method, Agrobacterium method and the like) accordingto the kind of the host.

Escherichia coli can be transformed according to the methods describedin, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17,107 (1982) and the like.

The genus Bacillus can be introduced into a vector according to themethods described in, for example, Molecular & General Genetics, 168,111 (1979) and the like.

A yeast can be introduced into a vector according to the methodsdescribed in, for example, Methods in Enzymology, 194, 182-187 (1991),Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

An insect cell and an insect can be introduced into a vector accordingto the methods described in, for example, Bio/Technology, 6, 47-55(1988) and the like.

An animal cell can be introduced into a vector according to the methodsdescribed in, for example, Cell Engineering additional volume 8, NewCell Engineering Experiment Protocol, 263-267 (1995) (published byShujunsha), and Virology, 52, 456 (1973).

A cell introduced with a vector can be cultured according to a knownmethod according to the kind of the host.

For example, when Escherichia coli or genus Bacillus is cultured, aliquid medium is preferable as a medium to be used for the culture. Themedium preferably contains a carbon source, nitrogen source, inorganicsubstance and the like necessary for the growth of the transformant.Examples of the carbon source include glucose, dextrin, soluble starch,sucrose and the like; examples of the nitrogen source include inorganicor organic substances such as ammonium salts, nitrate salts, corn steepliquor, peptone, casein, meat extract, soybean cake, potato extract andthe like; and examples of the inorganic substance include calciumchloride, sodium dihydrogen phosphate, magnesium chloride and the like.The medium may contain yeast extract, vitamins, growth promoting factorand the like. The pH of the medium is preferably about 5-about 8.

As a medium for culturing Escherichia coli, for example, M9 mediumcontaining glucose, casamino acid [Journal of Experiments in MolecularGenetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] ispreferable. Where necessary, for example, agents such as3β-indolylacrylic acid may be added to the medium to ensure an efficientfunction of a promoter. Escherichia coli is cultured at generally about15-about 43° C. Where necessary, aeration and stirring may be performed.

The genus Bacillus is cultured at generally about 30-about 40° C. Wherenecessary, aeration and stirring may be performed.

Examples of the medium for culturing yeast include Burkholder minimummedium [Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)], SD mediumcontaining 0.5% casamino acid [Proc. Natl. Acad. Sci. USA, 81, 5330(1984)] and the like. The pH of the medium is preferably about 5-about8. The culture is performed at generally about 20° C.-about 35° C. Wherenecessary, aeration and stirring may be performed.

As a medium for culturing an insect cell or insect, for example, Grace'sInsect Medium [Nature, 195, 788 (1962)] containing an additive such asinactivated 10% bovine serum and the like as appropriate and the likeare used. The pH of the medium is preferably about 6.2-about 6.4. Theculture is performed at generally about 27° C. Where necessary, aerationand stirring may be performed.

As a medium for culturing an animal cell, for example, minimum essentialmedium (MEM) containing about 5-about 20% of fetal bovine serum[Science, 122, 501 (1952)], Dulbecco's modified Eagle medium (DMEM)[Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the AmericanMedical Association, 199, 519 (1967)], 199 medium [Proceeding of theSociety for the Biological Medicine, 73, 1 (1950)] and the like areused. The pH of the medium is preferably about 6-about 8. The culture isperformed at generally about 30° C.-about 40° C. Where necessary,aeration and stirring may be performed.

As a medium for culturing a plant cell, for example, MS medium, LSmedium, B5 medium and the like are used. The pH of the medium ispreferably about 5-about 8. The culture is performed at generally about20° C.-about 30° C. Where necessary, aeration and stirring may beperformed.

As mentioned above, a complex of a nucleic acid sequence-recognizingmodule and a DNA glycosylase, i.e., nucleic acid-modifying enzymecomplex, can be expressed intracellularly.

An RNA encoding a nucleic acid sequence-recognizing module and/or a DNAglycosylase can be introduced into a host cell by microinjection method,lipofection method and the like. RNA introduction can be performed onceor repeated multiple times (e.g., 2-5 times) at suitable intervals.

When a complex of a nucleic acid sequence-recognizing module and a DNAglycosylase is expressed by an expression vector or RNA moleculeintroduced into the cell, the nucleic acid sequence-recognizing modulespecifically recognizes and binds to a target nucleotide sequence in thedouble stranded DNA (e.g., genomic DNA) of interest and, due to theaction of the DNA glycosylase linked to the nucleic acidsequence-recognizing module, base excision reaction occurs in the sensestrand or antisense strand of the targeted site (whole or partial targetnucleotide sequence or appropriately adjusted within several hundredbases including the vicinity thereof) and an abasic site (AP site) isproduced in one of the strands of the double stranded DNA. Then, thebase excision repair (BER) system in the cell operates, AP endonucleasefirst recognizes the AP site and cleaves the phosphoric acid bond in oneof DNA strand, and exonuclease removes nucleotide subjected to baseexcision. Then, DNA polymerase inserts a new nucleotide by using theopposing strand DNA as a template and finally DNA ligase repairs thejoint. Various mutations are introduced by a repair miss occurring atany stage of this BER. As mentioned above, the BER mechanism in the cellis inhibited, and the frequency of repair miss, and thus, efficiency ofmutation induction can be improved by using a mutant AP endonucleasewhich lost enzyme activity but retains binding capacity to AP site incombination.

As for zinc finger motif, production of many actually functionable zincfinger motifs is not easy, since production efficiency of a zinc fingerthat specifically binds to a target nucleotide sequence is not high andselection of a zinc finger having high binding specificity iscomplicated. While TAL effector and PPR motif have a high degree offreedom of target nucleic acid sequence recognition as compared to zincfinger motif, a problem remains in the efficiency since a large proteinneeds to be designed and constructed every time according to the targetnucleotide sequence. Furthermore, since these nucleic acidsequence-recognizing modules do not have a function to change thestructure of double stranded DNA (causing strain in the double helixstructure), for a DNA glycosylase with sufficiently low reactivity witha DNA having an unrelaxed double helix structure to efficiently act onthe targeted site, it is necessary to separately contact a factor thatchanges the structure of the double stranded DNA with the object doublestranded DNA, thus making the operation complicated.

In contrast, since the CRISPR-Cas system recognizes the object doublestranded DNA sequence by a guide RNA complementary to the targetnucleotide sequence, any sequence can be targeted by simply synthesizingan oligoDNA capable of specifically forming a hybrid with the targetnucleotide sequence. Moreover, at the targeted site, since the doublestranded DNA is unwound to generate a region having a single strandedstructure, and a region adjacent thereto which has a structure ofrelaxed double stranded DNA, DNA glycosylase can be made to actefficiently in a targeted site-specific manner, without combiningfactors that change the structure of double stranded DNA.

Therefore, in a more preferable embodiment of the present invention, aCRISPR-Cas system wherein at least one DNA cleavage ability of Cas isinactivated (CRISPR-mutant Cas), is used as a nucleic acidsequence-recognizing module.

FIG. 2 is a schematic showing of the double stranded DNA modificationmethod of the present invention using CRISPR-mutant Cas as a nucleicacid sequence-recognizing module.

The nucleic acid sequence-recognizing module of the present inventionusing CRISPR-mutant Cas is provided as a complex of an RNA moleculeconsisting of a guide RNA complementary to the target nucleotidesequence and tracrRNA necessary for recruiting mutant Cas protein, and amutant Cas protein.

The Cas protein to be used in the present invention is not particularlylimited as long as it belongs to the CRISPR system, and preferred isCas9. Examples of Cas9 include, but are not limited to, Streptococcuspyogenes-derived Cas9 (SpCas9), Streptococcus thermophilus-derived Cas9(StCas9) and the like. Preferred is SpCas9. AS a mutant Cas to be usedin the present invention, any of Cas wherein the cleavage ability of theboth strands of the double stranded DNA is inactivated and one havingnickase activity wherein at least one cleavage ability of one strandalone is inactivated can be used. For example, in the case of SpCas9, aD10A mutant in which the 10th Asp residue is converted to an Ala residueand lacking cleavage ability of a strand opposite to the strand forminga complementary strand with a guide RNA, or H840A mutant in which the840th His residue is converted to an Ala residue and lacking cleavageability of strand complementary to guide RNA, or a double mutant thereofcan be used, and other mutant Cas can be used similarly.

DNA glycosylase is provided as a complex with mutant Cas by a methodsimilar to the coupling scheme with the above-mentioned zinc finger andthe like. Alternatively, a DNA glycosylase and mutant Cas can also bebound by utilizing RNA aptamers MS2F6, PP7 and the like and RNA scaffoldby binding proteins thereto. Guide RNA forms a complementary strand withthe target nucleotide sequence, mutant Cas is recruited by the tracrRNAattached and mutant Cas recognizes DNA cleavage site recognitionsequence PAM (protospacer adjacent motif) (when SpCas9 is used, PAM is 3bases of NGG (N is any base), and, theoretically, can target anyposition on the genome). One or both DNAs cannot be cleaved, and, due tothe action of the DNA glycosylase linked to the mutant Cas, baseexcision occurs in the targeted site (appropriately adjusted withinseveral hundred bases including whole or partial target nucleotidesequence) and a AP site occurs in the double stranded DNA. Variousmutations are introduced due to the errors made by the BER system of thecell to be repaired (see, for example, FIG. 5).

Even when CRISPR-mutant Cas is used as a nucleic acidsequence-recognizing module, a nucleic acid sequence-recognizing moduleand a DNA glycosylase are desirably introduced, in the form of a nucleicacid encoding same, into a cell having a double stranded DNA ofinterest, similar to when zinc finger and the like are used as a nucleicacid sequence-recognizing module.

A DNA encoding Cas can be cloned by a method similar to theabove-mentioned method for a DNA encoding a DNA glycosylase, from a cellproducing the enzyme. A mutant Cas can be obtained by introducing amutation to convert an amino acid residue of the part important for theDNA cleavage activity (e.g., 10th Asp residue and 840th His residue forCas9, though not limited thereto) to other amino acid, into a DNAencoding cloned Cas, by a site specific mutation induction method knownper se.

Alternatively, a DNA encoding mutant Cas can also be constructed as aDNA showing codon usage suitable for expression in a host cell to beused, by a method similar to those mentioned above for a DNA encoding anucleic acid sequence-recognizing module and a DNA encoding a DNAglycosylase, and by a combination of chemical synthesis or PCR method orGibson Assembly method. For example, CDS sequence and amino acidsequence optimized for the expression of SpCas9 in eukaryotic cells areshown in SEQ ID NOs: 3 and 4. In the sequence shown in SEQ ID NO: 3,when “A” is converted to “C” in base No. 29, a DNA encoding a D10Amutant can be obtained, and when “CA” is converted to “GC” in base No.2518-2519, a DNA encoding an H840A mutant can be obtained.

A DNA encoding a mutant Cas and a DNA encoding a DNA glycosylase may belinked to allow for expression as a fusion protein, or designed to beseparately expressed using a binding domain, intein and the like, andform a complex in a host cell via protein-protein interaction andprotein ligation. Alternatively, a design may be employed in which a DNAencoding mutant Cas and a DNA encoding DNA glycosylase are each splitinto two fragments at suitable split site, either fragments are linkedto each other directly or via a suitable linker to express a nucleicacid-modifying enzyme complex as two partial complexes, which areassociated and refolded in the cell to reconstitute functional mutantCas having a particular nucleic acid sequence recognition ability, and afunctional DNA glycosylase having a base excision reaction catalystactivity is reconstituted when the mutant Cas is bonded to the targetnucleotide sequence. For example, a DNA encoding the N-terminal sidefragment and a DNA encoding the C-terminal side fragment of mutant Casare respectively prepared by the PCR method by using suitable primers; aDNA encoding the N-terminal side fragment and a DNA encoding theC-terminal side fragment of DNA glycosylase are prepared in the samemanner; for example, the DNAs encoding the N-terminal side fragments arelinked to each other, and the DNAs encoding the C-terminal sidefragments are linked to each other by a conventional method, whereby aDNA encoding two partial complexes can be produced. Alternatively, a DNAencoding the N-terminal side fragment of mutant Cas and a DNA encodingthe C-terminal side fragment of DNA glycosylase are linked; and a DNAencoding the N-terminal side fragment of DNA glycosylase and a DNAencoding the C-terminal side fragment of mutant Cas are linked, wherebya DNA encoding two partial complexes can also be produced. Respectivepartial complexes may be linked to allow for expression as a fusionprotein, or designed to be separately expressed using a binding domain,intein and the like, and foil a complex in a host cell viaprotein-protein interaction and protein ligation. Two partial complexesmay be linked to be expressed as a fusion protein. The split site of themutant Cas is not particularly limited as long as the two splitfragments can be reconstituted such that they recognize and bind to thetarget nucleotide sequence, and it may be split at one site to provideN-terminal side fragment and C-terminal side fragment, or not less than3 fragments obtained by splitting at two or more sites may beappropriately linked to give two fragments. The three-dimensionalstructures of various Cas proteins are known, and those of ordinaryskill in the art can appropriately select the split site based on suchinformation. For example, since the region consisting of the 94th to the718th amino acids from the N terminus of SpCas9 is a domain (REC)involved in the recognition of the target nucleotide sequence and guideRNA, and the region consisting of the 1099th amino acid to theC-terminal amino acid is the domain (PI) involved in the interactionwith PAM, the N-terminal side fragment and the C-terminal side fragmentcan be split at any site in REC domain or PI domain, preferably in aregion free of a structure (e.g., between 204th and 205th amino stepfrom the N-terminal (204 . . . 205), between 535th and 536th amino acidsfrom the N-terminal (535 . . . 536) and the like) (see, for example, NatBiotechnol. 33(2): 139-142 (2015)).

The obtained DNA encoding a mutant Cas and/or a DNA glycosylase can beinserted into the downstream of a promoter of an expression vectorsimilar to the one mentioned above, according to the host.

On the other hand, a DNA encoding guide RNA and tracrRNA can be obtainedby designing an oligoDNA sequence linking guide RNA sequencecomplementary to the target nucleotide sequence and known tracrRNAsequence (e.g.,gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtggtgctttt; SEQ ID NO: 5) and chemically synthesizing using aDNA/RNA synthesizer. While a DNA encoding guide RNA and tracrRNA canalso be inserted into an expression vector similar to the one mentionedabove, according to the host. As the promoter, pol III system promoter(e.g., SNR6, SNR52, SCR1, RPR1, U6, H1 promoter etc.) and terminator(e.g., T₆ sequence) are preferably used.

An RNA encoding mutant Cas and/or a DNA glycosylase can be prepared by,for example, transcription to mRNA in a vitro transcription system knownper se by using a vector encoding the above-mentioned mutant Cas and/orDNA encoding a DNA glycosylase as a template.

Guide RNA-tracrRNA can be obtained by designing an oligoDNA sequencelinking a sequence complementary to the target nucleotide sequence andknown tracrRNA sequence and chemically synthesizing using a DNA/RNAsynthesizer.

A DNA or RNA encoding mutant Cas and/or a DNA glycosylase, guideRNA-tracrRNA or a DNA encoding same can be introduced into a host cellby a method similar to the above, according to the host.

Since conventional artificial nuclease accompanies double stranded DNAbreaks (DSB), inhibition of growth and cell death assumedly caused bydisordered cleavage of chromosome (off-target cleavage) occur bytargeting a sequence in the genome. The effect thereof is particularlyfatal for many microorganisms and prokaryotes, and preventsapplicability. In the present invention, mutation is introduced not byDNA cleavage but by a base excision reaction on the DNA base, andtherefore, drastic reduction of toxicity can be realized.

The modification of the double stranded DNA in the present inventiondoes not prevent occurrence of cleavage of the double stranded DNA in asite other than the targeted site (appropriately adjusted within severalhundred bases including whole or partial target nucleotide sequence).However, one of the greatest advantages of the present invention isavoidance of toxicity by off-target cleavage, which is generallyapplicable to any species. In preferable one embodiment, therefore, themodification of the double stranded DNA in the present invention doesnot accompany cleavage of DNA strand not only in a targeted site of agiven double stranded DNA but in a site other than same.

As shown in the below-mentioned Examples, when sequence-recognizingmodules are produced corresponding to the adjacent multiple targetnucleotide sequences, and simultaneously used, the mutation introductioncan be efficiency increased than by using a single nucleotide sequenceas a target. As the effect thereof, similarly mutation induction isrealized even when both target nucleotide sequences partly overlap orwhen the both are apart by about 600 bp. It can occur both when thetarget nucleotide sequences are in the same direction (target nucleotidesequences are present on the same strand), and when they are opposed(target nucleotide sequence is present on each strand of double strandedDNA).

The genome sequence modification method of the present invention canintroduce mutation into almost all cells in which the nucleicacid-modifying enzyme complex of the present invention has beenexpressed, by selecting a suitable target nucleotide sequence. Thus,insertion and selection of a selection marker gene, which are essentialin the conventional genome editing, are not necessary. This dramaticallyfacilitates and simplifies gene manipulation and enlarges theapplicability to crop breeding and the like since a recombinant organismwith foreign DNA is not produced.

Since the genome sequence modification method of the present inventionshows extremely high efficiency of mutation induction, and does notrequire selection by markers, modification of multiple DNA regions atcompletely different positions as targets can be performed. Therefore,in one preferable embodiment of the present invention, two or more kindsof nucleic acid sequence-recognizing modules that specifically bind todifferent target nucleotide sequences (which may be present in oneobject gene, or two or more different object genes, which object genesmay be present on the same chromosome or different chromosomes) can beused. In this case, each one of these nucleic acid sequence-recognizingmodules and DNA glycosylase form a nucleic acid-modifying enzymecomplex. Here, a common DNA glycosylase can be used. For example, whenCRISPR-Cas system is used as a nucleic acid sequence-recognizing module,a common complex of a Cas protein and a DNA glycosylase (includingfusion protein) is used, and two or more kinds of chimeric RNAs oftracrRNA and each of two or more guide RNAs that respectively form acomplementary strand with a different target nucleotide sequence areproduced and used as guide RNA-tracrRNAs. On the other hand, when zincfinger motif, TAL effector and the like are used as nucleic acidsequence-recognizing modules, for example, a DNA glycosylase can befused with a nucleic acid sequence-recognizing module that specificallybinds to a different target nucleotide.

To express the nucleic acid-modifying enzyme complex of the presentinvention in a host cell, as mentioned above, an expression vectorcontaining a DNA encoding the nucleic acid-modifying enzyme complex, oran RNA encoding the nucleic acid-modifying enzyme complex is introducedinto a host cell. For efficient introduction of mutation, it isdesirable to maintain an expression of nucleic acid-modifying enzymecomplex of a given level or above for not less than a given period. Fromsuch aspect, it is ensuring to introduce an expression vector (plasmidetc.) autonomously replicatable in a host cell. However, since theplasmid etc. are foreign DNAs, they are preferably removed rapidly aftersuccessful introduction of mutation. When mutant AP endonuclease is usedin combination, since the mutant enzyme inhibits the BER mechanism inthe host cell, it may induce undesirable spontaneous mutations outsidethe target region. Thus, it is preferable to also remove a plasmidcontaining a DNA encoding the mutant enzyme promptly after introductionof the desired mutation. Therefore, though subject to change dependingon the kind of host cell and the like, for example, the introducedplasmid is desirably removed from the host cell after a lapse of for 6hr-2 days from the introduction of an expression vector by using variousplasmid removal methods well known in the art.

Alternatively, as long as expression of a nucleic acid-modifying enzymecomplex, which is sufficient for the introduction of mutation, isobtained, it is preferable to introduce mutation into the object doublestranded DNA by transient expression by using an expression vectorwithout autonomous replicatability in a host cell (e.g., vector etc.lacking replication origin that functions in host cell and/or geneencoding protein necessary for replication) or RNA.

The present invention is explained in the following by referring toExamples, which are not to be construed as limitative.

EXAMPLES

In the below-mentioned Examples, experiments were performed as follows.

<Cell Line, Culture, Transformation, and Expression Induction>

Budding yeast Saccharomyces cerevisiae BY4741 strain (requiring leucineand uracil) was cultured in a standard YPDA medium or SD medium with aDropout composition meeting the auxotrophicity. The culture performedwas stand culture in an agar plate or shaking culture in a liquid mediumbetween 25° C. and 30° C. Transformation was performed by an acetic acidlithium method, and selection was made in SD medium meeting appropriateauxotrophicity. For expression induction by galactose, after precultureovernight in an appropriate SD medium, culture in SR medium with carbonsource changed from 2% glucose to 2% raffinose overnight, and furtherculture in SGal medium with carbon source changed to 0.2% galactose for3 hr to 20 about two nights were conducted for expression induction.

For the measurement of the number of surviving cells and Can1 mutationrate, a cell suspension was appropriately diluted, applied on SD platemedium or SD-Arg+60 mg/l Canavanine plate medium or SD+300 mg/lCanavanine plate medium, applied, and the number of colonies that emerge3 days later was counted as the number of surviving cells. Using numberof surviving colonies in SD plate as the total number of cells, and thenumber of surviving colonies in Canavanine plate as resistant mutantstrain number, the mutation rate was calculated and evaluated. The siteof mutation introduction was identified by amplifying DNA fragmentscontaining the target gene region of each strain by a colony PCR method,performing DNA sequencing, and performing an alignment analysis based onthe sequence of Saccharomyces Genome Database (www.yeastgenome.org/).

<Nucleic Acid Operation>

DNA was processed or constructed by any of PCR method, restrictionenzyme treatment, ligation, Gibson Assembly method, and artificialchemical synthesis. For plasmid, as a yeast Escherichia coli shuttlevector, pRS315 for leucine selection and pRS426 for uracil selectionwere used as the backbone. Plasmid was amplified by Escherichia coliline XL-10 gold or DH5a, and introduced into yeast by the acetic acidlithium method.

<Construct>

For inducible expression, budding yeast pGal1/10 (SEQ ID NO: 6) which isa bidirectional promoter induced by galactose was used. At thedownstream of the promoter, a nuclear localization signal (ccc aag aagaag agg aag gtg; SEQ ID NO: 7 (PKKKRV; encoding SEQ ID NO: 8)) was addedto Streptococcus pyogenes-derived Cas9 gene ORF having a codon optimizedfor eucaryon expression (SEQ ID NO: 3) and the sequence of ORF (ORF ofwild-type gene is shown in SEQ ID NO: 1, Y164A mutation is substitutionof base number 490-491 ta with gc (ta490gc); Y164G mutation issubstitution of base number 490-491 ta with gg (ta490gg); N222D mutationis substitution of base number 664 a with g (a664g); L304A mutation issubstitution of base number 910-911 tt with gc (tt910gc); R308E mutationis substitution of base number 922-923 ag with ga (ag922ga); R3080mutation is substitution of base number 922-924 aga with tgt(aga922tgt)) of wild-type or various mutant uracil-DNA glycosylase genes(UNG1 derived from yeast Saccharomyces cerevisiae), excluding a region(base number 1-60) encoding the mitochondria localization signal, wasligated via a linker sequence and expressed as a fusion protein. Forcomparison, UNG1 gene instead of deaminase gene (PmCDA1 derived fromPetromyzon marinus Petromyzon marinus) was ligated and expressed as afusion protein. As a linker sequence, 2×GS linker (two repeats of ggtgga gga ggt tct; SEQ ID NO: 9 (GGGGS; encoding SEQ ID NO: 10)) was used.As a terminator, budding yeast-derived ADH1 terminator (SEQ ID NO: 11)and Top2 terminator (SEQ ID NO: 12) were ligated. In the domainintegration method, Cas9 gene ORF was ligated to SH3 domain (SEQ ID NOs:13 and 14) via 2×GS linker to give one protein, mutant yeast UNG1 addedwith SH3 ligand sequence (SEQ ID NOs: 15 and 16) as another protein andthey were ligated to Gal1/10 promoter on both directions andsimultaneously expressed. These were incorporated into pRS315 plasmid.

In Cas9, mutation to convert the 10th aspartic acid to alanine (D10A,corresponding DNA sequence mutation a29c) and mutation to convert the840th histidine to alanine (H840A, corresponding DNA sequence mutationca2518gc) were introduced to remove cleavage ability of each side of DNAstrand.

gRNA as a chimeric structure with tracrRNA (derived from Streptococcuspyogenes; SEQ ID NO: 5) was disposed between SNR52 promoter (SEQ ID NO:17) and Sup4 terminator (SEQ ID NO: 18), and incorporated into pRS426plasmid. As gRNA target base sequence, 793-812 (aacccaggtgcctggggtcc;SEQ ID NO: 19) and 767-786 complementary strand sequence(ataacggaatccaactgggc; SEQ ID NO: 20) of CAN1 gene ORF were used. Forsimultaneous expression of multiple targets, a sequence from a promoterto a terminator as one set and a plurality thereof were incorporatedinto the same plasmid. They were introduced into cells along withCas9-UNG1 expression plasmid, intracellularly expressed, and a complexof gRNA-tracrRNA and Cas9-UNG1 was formed.

Example 1: Modification of Genome Sequence by Linking DNA SequenceRecognition Ability of CRISPR-Cas to Mutant Uracil-DNA Glycosylase (1)

To test the effect of genome sequence modification technique of thepresent invention by utilizing mutant uracil-DNA glycosylase andCRISPR-Cas nucleic acid sequence recognition ability, introduction ofmutation into CAN1 gene encoding canavanine transporter that acquirecanavanine-resistance due to gene deficiency was tried. As gRNA, asequence complementary to 793-812 of CAN1 gene ORF and a sequencecomplementary to 767-786 complementary strand sequence were used, achimeric RNA expression vector obtained by linking thereto Streptococcuspyogenes-derived tracrRNA, and a vector expressing a protein obtained byfusing dCas9 with impaired nuclease activity by introducing mutations(D10A and H840A) into Streptococcus pyogenes-derived Cas9 (SpCas9), andwild-type yeast-derived UNG1 or yeast-derived UNG1 introduced withvarious mutations (N222D single mutation and double mutation of N222Dand L304A, R308E or R308C mutation) were constructed, introduced intothe budding yeast by the acetic acid lithium method, and coexpressed.The results are shown in FIG. 3. When cultured on acanavanine-containing SD plate, only the cells subjected to introductionand expression of gRNA-tracrRNA and dCas9-mutant UNG1 (double mutant ofN222D mutation imparting CDG activity and L304A, R308E or R3080 mutationthat decreases reactivity with DNA having an unrelaxed double helixstructure) formed canavanine-resistant colonies. With N222D singlemutation, the cytotoxicity was strong, and cell culture and evaluationwere difficult, and therefore, the results are not shown. From theabove, it was shown that target specific mutation introduction becomespossible by decreasing the reactivity of DNA glycosylase with a DNAhaving an unrelaxed double helix structure.

Example 2: Modification of Genome Sequence by Linking DNA SequenceRecognition Ability of CRISPR-Cas to Mutant Uracil-DNA Glycosylase (2)

Using yeast UNG1 introduced with double mutation of R308C mutation thatdecreases reactivity with a DNA having an unrelaxed double helixstructure, and N222D mutation imparting CDG activity, or Y164A or Y164Gmutation imparting TDG activity, and by a method similar to that inExample 1, introduction of mutation into CAN1 gene was tried. Theresults are shown in FIG. 4. It was shown that R3080 N222D can achieveefficiency of mutation induction comparable to that of deaminase PmCDA1,and mutant strain can be obtained even without selection. It was shownthat thymine base could be edited, since canavanine-resistant colony wasalso obtained in R3080 Y164A. Y164G mutation improved the efficiency ofmutation induction.

Then, each canavanine-resistant clone was subjected to the sequenceanalysis of the Can1 gene region. The results are shown in FIG. 5.Mutations were somewhat randomly centered around two adjacent targetsites (767-786, 793-812). This is different from the pinpointintroduction of mutation by deaminase (WO 2015-133554), and suggeststhat the genome editing technique of the present invention is suitablefor random introduction of mutation into the target nucleotide sequenceand in the vicinity thereof. As assumed, point mutation from C or G wasmainly found in N222D, and point mutation from T or A was mainly foundin Y164A and Y164G.

Example 3: Use of Different Coupling Scheme

Whether mutation can be introduced into a targeted gene even when Cas9and DNA glycosylase are not used as a fusion protein but when a nucleicacid-modifying enzyme complex is formed via a binding domain and aligand thereof was examined. As Cas9, dCas9 used in Example 1 was used,yeast UNG1 mutant (double mutant of N222D or Y164A mutation, and R308Eor R308C mutant) was used as DNA glycosylase, SH3 domain was fused withthe former, and a binding ligand thereof was fused with the latter toproduce various constructs shown in FIG. 6. In the same manner as inExample 1, sequences in the CAN1 gene were used as gRNA targets, andthese constructs were introduced into a budding yeast. As a result, evenwhen dCas9 and DNA glycosylase were bound via the binding domain,mutation was efficiently introduced into the targeted site of the CAN1gene (FIG. 6).

Example 4 Improvement of Efficiency of Mutation Induction byCoexpression of Mutant AP Endonuclease

Using yeast UNG1 introduced with double mutation of R308C mutation thatdecreases reactivity with a DNA having an unrelaxed double helixstructure, and N222D mutation imparting CDG activity or Y164G mutationimparting TDG activity, and mutant human APE1 (E96Q, D210N) which lostenzyme activity but retained binding capacity to AP site, and by amethod similar to that in Example 1, introduction of mutation into CAN1gene was tried. The results are shown in FIG. 7. When mutant APE1 wascoexpressed, the number of canavanine-resistant colonies increased evenin Y164G, R3080 that showed low efficiency when used alone, andefficiency of mutation introduction targeting thymine was remarkablyimproved.

Example 5 Reduction of Cytotoxicity by Introduction of Mutation thatDecreases Reactivity with DNa Having an Unrelaxed Double Helix Structure

An influence of the presence or absence of mutation (L304A) thatdecreases reactivity with a DNA having an unrelaxed double helixstructure in UNG1 on the survival rate of the host yeast was examined.The results are shown in FIG. 8. The host yeast introduced with mutantUNG1 having only the mutation imparting CDG activity (N222D) or TDGactivity (Y164A) showed a marked decrease in the survival rate ascompared to the yeast introduced with wild-type UNG1. This is assumed tobe because wild-type UNG1 removes uracil which is an aberrant base thatappears rarely in DNA, whereas mutant UNG1 having CDG activity or TDGactivity removes cytosine or thymine anywhere on the genomic DNA andproduces mutations undesirable for the survival of the cell. On theother hand, when the reactivity with a DNA having an unrelaxed doublehelix structure is decreased by introducing L304 mutation, the survivalrate of the host yeast recovered remarkably and cytotoxicity could beavoided.

Example 6 Utilization of Heterogenous Uracil-DNA Glycosylase

Whether introduction of targeted mutation into the host yeast ispossible even when heterogenous mutant UNG1 is used instead of mutantUNG1 derived from yeast was examined. Two kinds of Escherichiacoli-derived mutant ungs (EcUDG) (N123D/L191A double mutant, Y66G/L191Adouble mutant) and four kinds of vaccinia virus-derived mutant UDGs(vvUDG) (N120D/R187C double mutant, Y70G/R187C double mutant, N120Dmutant, Y70G mutant) were used. The results are shown in FIG. 9. Whileboth EcUDG, vvUDG were functional in yeast, the efficiency of mutationinduction was low as compared to yeast UNG1, and it was shown that theuse of allogeneic DNA glycosylase was advantageous. Surprisingly, it wasclarified that cytotoxicity was absent in vvUDG, regardless of thepresence or absence of R187C mutation corresponding to R3080 mutation ofyeast UNG1. As a result of sequence analysis, since mutation by vvUDGwas concentrated in a specific base in the target nucleotide sequenceregardless of the presence or absence of R187C mutation (see FIG. 10),vvUDG was suggested to be a DNA glycosylase with natively sufficientlylow reactivity with a DNA having an unrelaxed double helix structure.The efficiency of mutation induction by vvUDG was remarkably increasedin virus DNA polymerase by coexpressing A20, which interacts with vvUDGand acts as a processivity factor (FIG. 9).

Example 7 Reduction of Non-Specific Mutation by Utilization of SplitEnzyme

In addition to the utilization of mutation that decreases reactivitywith a DNA having an unrelaxed double helix structure, and DNAglycosylase with natively low reactivity with a DNA having a doublehelix structure such as vvUDG, utilization of split enzyme technique wastried as a different means for reducing non-specific mutation by DNAglycosylase. The plasmids shown in FIG. 11 containing DNA encodingvarious split enzymes were introduced into the host yeast together witha plasmid containing a DNA encoding guide RNA-tracrRNA by a methodsimilar to that in Example 1, and the cell number, the number ofcanavanine-resistant (mutation at targeted site) colonies, the number ofthialysine-resistant (non-specific mutation) colonies on a nonselectivemedium were examined. The survival rate of the host yeast on anonselective medium was equivalent to that when mutant UNG1 introducedwith mutation (R3080) that decreases reactivity with a DNA having anunrelaxed double helix structure was introduced even when any splitenzyme was used. Thus, it was shown that cytotoxicity can besufficiently decreased by the utilization of a split enzyme, evenwithout introducing a mutation that decreases reactivity with a DNAhaving an unrelaxed double helix structure, whereby it was suggestedthat non-specific mutation can be suppressed (FIG. 11). In fact, thefrequency of non-specific mutation by using thialysine-resistance as anindex was decreased by the utilization of a split enzyme (FIG. 11).

INDUSTRIAL APPLICABILITY

The present invention makes it possible to safely introduce sitespecific mutation into any species without accompanying insertion of aforeign DNA or double-stranded DNA breaks. It is also possible to set awide range of mutation introduction to target nucleotide sequence andseveral hundred bases in the vicinity thereof, and the technique canalso be applied to topical evolution induction by introduction of randommutation into a particular restricted region, which has been almostimpossible heretofore, and is extremely useful. Furthermore, whenmutation imparting CDG activity and mutation imparting TDG activity areimparted to UNG, base excision using 5-methylcytidine as a substratebecomes possible. According to the present invention, therefore, theepigenome information can be rewritten into, for example,region-specific release of methylation state to change the geneexpression pattern and the like. Therefore, artificial celldifferentiation, cancer cell inhibition, modification of gene functionwithout rewriting genome sequence, and the like become possible.

This application is based on a patent application No. 2014-224745 filedin Japan (filing date: Nov. 4, 2014), the contents of which areincorporated in full herein.

The invention claimed is:
 1. A method of modifying a targeted site of adouble stranded DNA in a eukaryotic cell, comprising contacting acomplex with the double stranded DNA, wherein the complex comprises anucleic acid sequence-recognizing module and a mutant of uracil DNAglycosylase having reduced reactivity with unrelaxed DNA to avoidcytotoxicity, wherein the nucleic acid sequence-recognizing modulespecifically binds to a target nucleotide sequence in the targeted siteof the double stranded DNA, wherein the mutant of uracil DNA glycosylaseis (i-a) a mutant of yeast UNG1 having N222D/L304A mutations,N222D/R308E mutations, N222D/R308C mutations, Y164A/L304A mutations,Y164A/R308E mutations, Y164A/R308C mutations, Y164G/L304A mutations,Y164G/R308E mutations, Y164G/R308C mutations, N222D/Y164A/L304Amutations, N222D/Y164A/R308E mutations, N222D/Y164A/R308C mutations,N222D/Y164G/L304A mutations, N222D/Y164G/R308E mutations orN222D/Y164G/R308C mutations, or (i-b) a mutant of Uracil DNA Glycosylase(UNG) other than a mutant of (i-a) and having mutations corresponding tothe mutations of the mutant of (i-a), thereby converting one or morenucleotides in the targeted site to other one or more nucleotides ordeleting one or more nucleotides, or inserting one or more nucleotidesinto said targeted site, without a double stranded DNA break in thetargeted site.
 2. The method according to claim 1, wherein the nucleicacid sequence-recognizing module is selected from the group consistingof a clustered regularly interspaced short palindromic repeats(CRISPR)-associated (CRISPR-Cas) system wherein at least one DNAcleavage ability of Cas nuclease is inactivated, a zinc finger motif, atranscription activator-like effector and a pentatricopeptide repeatmotif.
 3. The method according to claim 1, wherein the nucleic acidsequence-recognizing module is a CRISPR-Cas system wherein at least oneDNA cleavage ability of Cas nuclease is inactivated.
 4. The methodaccording to claim 3, wherein the Cas nuclease is Cas9 nuclease.
 5. Themethod according to claim 1, wherein the double stranded DNA is furthercontacted with a factor that changes a DNA double stranded structure. 6.The method according to claim 1, which uses two or more kinds of nucleicacid sequence-recognizing modules each specifically binding to adifferent target nucleotide sequence.
 7. The method according to claim6, wherein the different target nucleotide sequences are present indifferent genes.
 8. The method according to claim 1, further comprisingcontacting the double stranded DNA with a mutant ofapurinic/apyrimidinic endonuclease having binding capacity to an abasicsite but lacking nuclease activity.
 9. The method according to claim 1,wherein the double stranded DNA is contacted with the complex byintroducing a nucleic acid comprising a sequence encoding the nucleicacid sequence-recognizing module and a sequence encoding the mutant ofuracil DNA glycosylase into a eukaryotic cell having the double strandedDNA.
 10. The method according to claim 9, wherein the cell is amicrobial cell.
 11. The method according to claim 9, wherein the cell isa plant cell, an insect cell, or an animal cell.
 12. The methodaccording to claim 11, wherein the animal cell is a vertebrate cell. 13.The method according to claim 12, wherein the vertebrate cell is amammalian cell.
 14. The method according to claim 9, wherein the cell isa polyploid cell, and all of the targeted sites in alleles on ahomologous chromosome are modified.
 15. The method according to claim 1,wherein the mutant of UNG of (i-b) is a mutant of ung derived fromEscherichia coli, mutant of UNG2 derived from yeast, or mutant of UNG1or UNG2 derived from human, mouse, swine, bovine, horse or monkey. 16.The method according to claim 1, wherein the complex is formed via aninteraction of a protein binding domain fused to the nucleic acidsequence-recognizing module and a binding partner of the domain fused tothe mutant of uracil DNA glycosylase.
 17. The method according to claim1, wherein the mutant of uracil DNA glycosylase is (i-a) a mutant ofyeast UNG1 having N222D/R308C mutations or N222D/Y164G/R308C mutations,or (i-b) a mutant of Uracil DNA Glycosylase (UNG) other than a mutant of(i-a) and having mutations corresponding to the mutations of the mutantof (i-a).
 18. A method of modifying a targeted site of a double strandedDNA in a eukaryotic cell, comprising contacting a complex with thedouble stranded DNA, wherein the complex comprises a nucleic acidsequence-recognizing module and a mutant of uracil DNA glycosylasehaving reduced reactivity with unrelaxed DNA to avoid cytotoxicity,wherein the nucleic acid sequence-recognizing module specifically bindsto a target nucleotide sequence in the targeted site of the doublestranded DNA, wherein the mutant of uracil DNA glycosylase is (i-a) amutant of yeast UNG1 having N222D/L304A mutations, N222D/R308Emutations, N222D/R308C mutations, Y164A/L304A mutations, Y164A/R308Emutations, Y164A/R308C mutations, Y164G/L304A mutations, Y164G/R308Emutations, Y164G/R308C mutations, N222D/Y164A/L304A mutations,N222D/Y164A/R308E mutations, N222D/Y164A/R308C mutations,N222D/Y164G/L304A mutations, N222D/Y164G/R308E mutations orN222D/Y164G/R308C mutations, or (i-b) or a mutant of Uracil DNAGlycosylase (UNG) other than a mutant of (i-a) and having mutationscorresponding to the mutations of the mutant of (i-a), wherein themutant of uracil DNA glycosylase, and an element of the nucleic acidsequence-recognizing module which is directly bonded to the mutant ofuracil DNA glycosylase are respectively split into two fragments, thefragments of either of the mutant of uracil DNA glycosylase and theelement are respectively linked to the fragments of the other to providetwo partial complexes, and when the partial complexes are refolded witheach other, the nucleic acid sequence-recognizing module is capable ofspecifically binding to the target nucleotide sequence and the specificbond enables the mutant of uracil DNA glycosylase to exhibit enzymeactivity.
 19. The method according to claim 18, wherein the element ofthe nucleic acid sequence-recognizing module which is directly bonded tothe mutant of uracil DNA glycosylase is a mutant of Cas nuclease whereinat least one of the DNA cleavage abilities is inactivated.
 20. Themethod according to claim 19, wherein the Cas nuclease is Cas9 nuclease.21. The method according to claim 18, wherein the two partial complexesare provided as separate molecule complexes, and are refolded byassociation thereof in the cell.
 22. The method according to claim 18,wherein the mutant of uracil DNA glycosylase is (i-a) a mutant of yeastUNG1 having N222D/R308C mutations or N222D/Y164G/R308C mutations, or(i-b) a mutant of Uracil DNA Glycosylase (UNG) other than a mutant of(i-a) and having mutations corresponding to the mutations of the mutantof (i-a).
 23. A nucleic acid-modifying enzyme complex comprising (a) anucleic acid sequence-recognizing module and (b) a mutant of uracil DNAglycosylase having reduced reactivity with unrelaxed DNA to avoidcytotoxicity, wherein the nucleic acid sequence-recognizing modulespecifically binds to a target nucleotide sequence in the targeted siteof the double stranded DNA, wherein the mutant of uracil DNA glycosylaseis (i-a) a mutant of yeast UNG1 having N222D/L304A mutations,N222D/R308E mutations, N222D/R308C mutations, Y164A/L304A mutations,Y164A/R308E mutations, Y164A/R308C mutations, Y164G/L304A mutations,Y164G/R308E mutations, Y164G/R308C mutations, N222D/Y164A/L304Amutations, N222D/Y164A/R308E mutations, N222D/Y164A/R308C mutations,N222D/Y164G/L304A mutations, N222D/Y164G/R308E mutations orN222D/Y164G/R308C mutations, or (i-b) a mutant of Uracil DNA Glycosylase(UNG) other than a mutant of (i-a) and having mutations corresponding tothe mutations of the mutant of (i-a).
 24. The complex according to claim23, wherein the nucleic acid sequence-recognizing module is selectedfrom the group consisting of a clustered regularly interspaced shortpalindromic repeats (CRISPR)-CRISPR associated system wherein at leastone DNA cleavage ability of Cas nuclease is inactivated, a zinc fingermotif, a transcription activator-like effector and a pentatricopeptiderepeat motif.
 25. The nucleic acid-modifying enzyme complex according toclaim 23, wherein the mutant of uracil DNA glycosylase is (i-a) a mutantof yeast UNG1 having N222D/R308C mutations or N222D/Y164G/R308Cmutations, or (i-b) a mutant of Uracil DNA Glycosylase (UNG) other thana mutant of (i-a) and having mutations corresponding to the mutations ofthe mutant of (i-a).
 26. A nucleic acid comprising a sequence encoding anucleic acid sequence-recognizing module, and a sequence encoding amutant of uracil DNA glycosylase having reduced reactivity withunrelaxed DNA to avoid cytotoxicity, wherein the nucleic acidsequence-recognizing module specifically binds to a target nucleotidesequence in the targeted site of the double stranded DNA, wherein themutant of uracil DNA glycosylase is (i-a) a mutant of yeast UNG1 havingN222D/L304A mutations, N222D/R308E mutations, N222D/R308C mutations,Y164A/L304A mutations, Y164A/R308E mutations, Y164A/R308C mutations,Y164G/L304A mutations, Y164G/R308E mutations, Y164G/R308C mutations,N222D/Y164A/L304A mutations, N222D/Y164A/R308E mutations,N222D/Y164A/R308C mutations, N222D/Y164G/L304A mutations,N222D/Y164G/R308E mutations or N222D/Y164G/R308C mutations, or (i-b) amutant of Uracil DNA Glycosylase (UNG) other than a mutant of (i-a) andhaving mutations corresponding to the mutations of the mutant of (i-a).27. The nucleic acid according to claim 26, wherein the nucleic acidsequence-recognizing module is selected from the group consisting of aclustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR associated system wherein at least one DNA cleavageability of Cas nuclease is inactivated, a zinc finger motif, atranscription activator-like effector and a pentatricopeptide repeatmotif.
 28. The nucleic acid according to claim 26, wherein the mutant ofuracil DNA glycosylase is (i-a) a mutant of yeast UNG1 havingN222D/R308C mutations or N222D/Y164G/R308C mutations, or (i-b) a mutantof Uracil DNA Glycosylase (UNG) other than a mutant of (i-a) and havingmutations corresponding to the mutations of the mutant of (i-a).